Romidepsin solid forms and uses thereof

ABSTRACT

The present disclosure provides solid forms of a compound of formula I. In some embodiments, the present disclosure provides crystalline forms of Compound I. In some embodiments, the present disclosure provides solvate forms of Compound I. In some embodiments, the present disclosure provides amorphous Compound I.

This application claims priority to U.S. Provisional Application No.61/363,522, filed Jul. 12, 2010, which is incorporated herewith byreference in its entirety.

FIELD

Provided herein are solid forms of romidepsin and compositionscomprising these forms. In some embodiments, provided are polymorphicforms of romidepsin. In some embodiments, provided are solvate forms ofromidepsin. In some embodiments, provided is amorphous romidepsin. Alsoprovided are methods for producing such forms and compositions.

BACKGROUND

Romidepsin is a natural product which was isolated from Chromobacteriumviolaceum by Fujisawa Pharmaceuticals. See Published Japanese PatentApplication Hei 7 (1995)-64872; and U.S. Pat. No. 4,977,138, issued Dec.11, 1990, each of which is incorporated herein by reference. Variouspreparations and purifications of romidepsin are described in PCTPublication WO 02/20817, which is incorporated herein by reference.

It is a bicyclic peptide consisting of four amino acid residues(D-valine, D-cysteine, dehydrobutyrine, and L-valine) and a novel acid(3-hydroxy-7-mercapto-4-heptenoic acid). Romidepsin is a depsipeptidewhich contains both amide and ester bonds. In addition to the productionof C. violaceum using fermentation, romidepsin can also be prepared bysynthetic or semi-synthetic means. The total synthesis of romidepsinreported by Kahn et al. (J. Am. Chem. Soc. 118:7237-7238, 1996) involves14 steps and yields romidepsin in 18% overall yield. The structure ofromidepsin is shown below and referred to hereinafter as “Compound I”:

Compound I has been shown to have anti-microbial, immunosuppressive, andanti-tumor activities. Compound I is approved in the U.S. for treatmentof cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma(PTCL), and is currently being tested, for example, for use in treatingpatients with other hematological malignancies (e.g., myeloma, etc.) andsolid tumors (e.g., prostate cancer, pancreatic cancer, etc.). It isthought to act by selectively inhibiting deacetylases (e.g., histonedeacetylase, tubulin deacetylase), promising new targets for thedevelopment of a new class of anti-cancer therapies (Nakajima et al.,Experimental Cell Res. 241:126-133, 1998). One mode of action involvesthe inhibition of one or more classes of histone deacetylases (HDAC).

SUMMARY

In one aspect, provided herein are solid forms of Compound I.

In some embodiments, provided herein is a method of preparation ofcrystalline form C of Compound I and its characterization.

In some embodiments, provided herein is a method of preparation ofcrystalline form D of Compound I and its characterization.

In some embodiments, provided herein is a method of preparation ofcrystalline form E of Compound I and its characterization.

In some embodiments, provided herein is a method of preparation ofcrystalline form I of Compound I and its characterization.

In some embodiments, provided herein is a method of preparation ofcrystalline form J of Compound I and its characterization.

In some embodiments, provided herein is a method of preparation ofcrystalline form K of Compound I and its characterization.

In some embodiments, provided herein is a method of preparation ofcrystalline form L of Compound I and its characterization.

In some embodiments, provided herein is a method of preparation ofcrystalline form N of Compound I and its characterization.

In some embodiments, provided herein is a method of preparation ofamorphous Compound I and its characterization.

In some embodiments, Compound I, and solid forms thereof, are used forthe preparation of pharmaceutical compositions. In some embodiments,provided are compositions and formulations (e.g., pharmaceuticalcompositions and formulations) comprising solid forms of Compound I.

In another aspect, provided herein are methods to treat proliferativediseases, immune-mediated diseases, infectious diseases, certaincirculatory diseases, and certain neurodegenerative diseases usingCompound I, its solid forms and compositions comprising same. In someembodiments, provided herein are methods to treat cancer. In someembodiments, cancers include, but are not limited to, carcinomas,sarcomas, leukemias, lymphomas and the like. In certain embodiments,cancer is a hematological malignancy. In certain embodiments, cancer isa solid tumor.

In another aspect, provided herein are methods of electrolytesupplementation for patients receiving Compound I therapy.

DESCRIPTION OF THE DRAWINGS

FIG. 1( a) depicts a representative solution ¹HNMR spectrum obtained forCompound I.

FIG. 1( b) depicts a molecular structure for Compound I.

FIG. 1( c) depicts an XRPD for Compound I Form C collected at roomtemperature.

FIG. 1( d) tabulates observed peaks (part i); and prominent peaks (partii) present in the XRPD of FIG. 1( c).

FIG. 1( e) depicts a DSC thermogram obtained for Compound I Form C.

FIG. 1( f) depicts a TGA thermogram obtained for Compound I Form C.

FIG. 1( g) depicts an FT-IR spectrum obtained for Compound I Form C.

FIG. 1( h) tabulates peak positions of bands present in the FT-IRspectrum of FIG. 1( g).

FIG. 1( i) depicts a calculated XRPD for Compound I Form C collected atsubambient temperature.

FIG. 1( j) depicts theoretical observed peaks (part i); andrepresentative peaks (part ii) present in the XRPD of FIG. 1( i).

FIG. 1( k) depicts an ORTEP drawing of Compound I, Form C, watermolecules not shown.

FIG. 1( l) depicts a packing diagram of Compound I, Form C viewed downthe crystallographic a axis.

FIG. 1( m) depicts a packing diagram of Compound I, Form C viewed downthe crystallographic b axis.

FIG. 1( n) depicts a packing diagram of Compound I, Form C viewed downthe crystallographic c axis.

FIG. 1( o) tabulates positional parameters and estimated standarddeviations for Compound I, Form C.

FIG. 1( p) tabulates bond distances (Angstroms) for Compound I, Form C.

FIG. 1( q) tabulates bond angles (degrees) for Compound I, Form C.

FIG. 2( a) depicts an XRPD for Compound I Form D collected at roomtemperature.

FIG. 2( b) tabulates observed peaks (part i); and prominent peaks (partii) present in the XRPD of FIG. 2( a).

FIG. 2( c) depicts a DSC thermogram obtained for Compound I Form D.

FIG. 2( d) depicts a TGA thermogram obtained for Compound I Form D.

FIG. 2( e) depicts an FT-IR spectrum obtained for Compound I Form D.

FIG. 2( f) tabulates peak positions of bands present in the FT-IRspectrum of FIG. 2( e).

FIG. 3( a) depicts an XRPD for Compound I Form E collected at roomtemperature.

FIG. 3( b) tabulates observed peaks (part i); and prominent peaks (partii) present in the XRPD of FIG. 3( a).

FIG. 3( c) depicts a DSC thermogram obtained for Compound I Form E.

FIG. 3( d) depicts a TGA thermogram obtained for Compound I Form E.

FIG. 3( e) depicts an FT-IR spectrum obtained for Compound I Form E.

FIG. 3( f) tabulates peak positions of bands present in the FT-IRspectrum of FIG. 3( e).

FIG. 3( g) depicts an FT-Raman spectrum for Compound I Form E.

FIG. 3( h) depicts a calculated XRPD for Compound I Form E collected atsubambient temperature.

FIG. 3( i) depicts theoretical observed peaks (part i); andrepresentative peaks (part ii) present in the XRPD of FIG. 3( h).

FIG. 3( j) depicts an ORTEP drawing of Compound I, Form E.

FIG. 3( k) depicts a packing diagram of Compound I, Form E viewed downthe crystallographic a axis.

FIG. 3( l) depicts a packing diagram of Compound I, Form E viewed downthe crystallographic b axis.

FIG. 3( m) depicts a packing diagram of Compound I, Form E viewed downthe crystallographic c axis.

FIG. 3( n) tabulates positional parameters and estimated standarddeviations for Compound I, Form E.

FIG. 3( o) tabulates bond distances (Angstroms) for Compound I, Form E.

FIG. 3( p) tabulates bond angles (degrees) for Compound I, Form E.

FIG. 4( a) depicts an XRPD for Compound I Form H collected at roomtemperature.

FIG. 4( b) tabulates observed peaks (part i); and prominent peaks (partii) present in the XRPD of FIG. 4( a).

FIG. 4( c) depicts a DSC thermogram obtained for Compound I Form H.

FIG. 4( d)) depicts a TGA thermogram obtained for Compound I Form H.

FIG. 4( e) depicts an FT-IR spectrum obtained for Compound I Form H.

FIG. 4( f) tabulates peak positions of bands present in the FT-IRspectrum of FIG. 4( e).

FIG. 5( a) depicts an XRPD for Compound I Form I collected at roomtemperature.

FIG. 5( b) tabulates observed peaks present in the XRPD of FIG. 5( a).

FIG. 5( c) depicts a DSC thermogram obtained for Compound I Form I.

FIG. 5( d) depicts a TGA thermogram obtained for Compound I Form I.

FIG. 5( e) depicts an FT-IR spectrum obtained for Compound I Form I.

FIG. 5( f) tabulates peak positions of bands present in the FT-IRspectrum of FIG. 5( e).

FIG. 5( g) depicts a calculated XRPD for Compound I Form I collected atsubambient temperature.

FIG. 5( h) depicts theoretical observed peaks (part i); andrepresentative peaks (part ii) present in the XRPD of FIG. 5( g).

FIG. 5( i) depicts an ORTEP drawing of Compound I, Form I, chloroformnot shown.

FIG. 5( j) depicts a packing diagram of Compound I, Form I viewed downthe crystallographic a axis.

FIG. 5( k) depicts a packing diagram of Compound I, Form I viewed downthe crystallographic b axis.

FIG. 5( l) depicts a packing diagram of Compound I, Form I viewed downthe crystallographic c axis.

FIG. 5( m) tabulates positional parameters and estimated standarddeviations for Compound I, Form I.

FIG. 5( n) tabulates bond distances (Angstroms) for Compound I, Form I.

FIG. 5( o) tabulates bond angles (degrees) for Compound I, Form I.

FIG. 5( p) depicts an XRPD for Compound I Form I.

FIG. 5( q) tabulates observed peaks present in the XRPD of FIG. 5( p).

FIG. 5( r) tabulates prominent peaks present in the XRPD of FIG. 5( p).

FIG. 5( s) depicts an FT-IR spectrum obtained for Compound I Form I.

FIG. 5( t) tabulates peak positions of bands present in the FT-IRspectrum of FIG. 5( s).

FIG. 5( u) depicts Panalytical X-Pert Pro MPD PW3040 data for Compound IForm I.

FIG. 5( v) depicts a DSC thermogram obtained for Compound I Form I.

FIG. 5( w) depicts a DSC thermogram obtained for Compound I Form I.

FIG. 5( x) depicts a TGA thermogram obtained for Compound I Form I.

FIG. 5( y) depicts an FT-IR spectrum obtained for Compound I Form I.

FIG. 6( a) depicts an X-ray diffraction pattern overlay of Compound IForm D and the calculated X-ray diffraction pattern of Compound I FormJ.

FIG. 6( b) depicts an ORTEP drawing of the single crystal structure ofCompound I Form J.

FIG. 6( c) depicts a calculated XRPD for Compound I Form J collected atsubambient temperature.

FIG. 6( d) depicts theoretical observed peaks (part i); and prominentpeaks (part ii) present in the XRPD of FIG. 6( c).

FIG. 6( e) depicts a packing diagram of Compound I, Form J viewed downthe crystallographic a axis.

FIG. 6( f) depicts a packing diagram of Compound I, Form J viewed downthe crystallographic b axis.

FIG. 6( g) depicts a packing diagram of Compound I, Form J viewed downthe crystallographic c axis.

FIG. 6( h) tabulates positional parameters and estimated standarddeviations for Compound I, Form J.

FIG. 6( i) tabulates bond distances (Angstroms) for Compound I, Form J.

FIG. 6( j) tabulates bond angles (degrees) for Compound I, Form J.

FIG. 6( k) depicts an XRPD for Compound I Form J.

FIG. 6( l) tabulates observed peaks present in the XRPD of FIG. 6( k).

FIG. 6( m) tabulates prominent peaks present in the XRPD of FIG. 6( k).

FIG. 6( n) depicts an FT-IR spectrum obtained for Compound I Form J.

FIG. 6( o) tabulates peak positions of bands present in the FT-IRspectrum of FIG. 6( n).

FIG. 6( p) depicts Panalytical X-Pert Pro MPD PW3040 data for Compound IForm J.

FIG. 6( q) depicts a DSC thermogram obtained for Compound I Form J.

FIG. 6( r) depicts a TGA thermogram obtained for Compound I Form J.

FIG. 6( s) depicts an FT-IR spectrum obtained for Compound I Form J.

FIG. 7( a) depicts an XRPD for amorphous Compound I collected at roomtemperature.

FIG. 7( b) depicts a modulated DSC thermogram obtained for amorphousCompound I.

FIG. 7( c) depicts a TGA thermogram obtained for amorphous Compound I.

FIG. 7( d) depicts an FT-IR spectrum obtained for amorphous Compound I.

FIG. 7( e) tabulates peak positions of bands present in the FT-IRspectrum of FIG. 7( d).

FIG. 7( f) depicts an FT-Raman spectrum for amorphous Compound I.

FIG. 8( a) depicts an XRPD for Compound I, Form K collected at roomtemperature.

FIG. 8( b) tabulates observed peaks (part i); and prominent peaks (partii); present in the XRPD of FIG. 8( a).

FIG. 8( c) depicts an XRPD for Compound I, Form K.

FIG. 8( d) tabulates observed peaks present in the XRPD of FIG. 8( c).

FIG. 8( e) tabulates prominent peaks present in the XRPD of FIG. 8( c).

FIG. 8( f) depicts an FT-IR spectrum obtained for Compound I Form K.

FIG. 8( g) tabulates peak positions of bands present in the FT-IRspectrum of FIG. 8( f).

FIG. 8( h) depicts Panalytical X-Pert Pro MPD PW3040 data for Compound IForm K.

FIG. 8( i) depicts a DSC thermogram obtained for Compound I Form K.

FIG. 8( j) depicts a DSC thermogram obtained for Compound I Form K.

FIG. 8( k) depicts a TGA thermogram obtained for Compound I Form K.

FIG. 8( l) depicts data for Compound I Form K.

FIG. 9( a) depicts an XRPD for Compound I Form F.

FIG. 9( b) tabulates observed peaks present in the XRPD of FIG. 9( a).

FIG. 9( c) tabulates prominent peaks present in the XRPD of FIG. 9( a).

FIG. 9( d) depicts an XRPD for Compound I Form F.

FIG. 9( e) tabulates observed peaks present in the XRPD of FIG. 9( d).

FIG. 9( f) tabulates prominent peaks present in the XRPD of FIG. 9( d).

FIG. 9( g) depicts an FT-IR spectrum obtained for Compound I Form F.

FIG. 9( h)) tabulates peak positions of bands present in the FT-IRspectrum of FIG. 9( g).

FIG. 9( i) depicts Panalytical X-Pert Pro MPD PW3040 data for Compound IForm F.

FIG. 9( j) depicts a DSC thermogram obtained for Compound I Form F.

FIG. 9( k) depicts a TGA thermogram obtained for Compound I Form F.

FIG. 9( l) depicts an FT-IR spectrum obtained for Compound I Form F.

FIG. 10( a) depicts an XRPD for Compound I Form L.

FIG. 10( b) tabulates observed peaks present in the XRPD of FIG. 10( a).

FIG. 10( c) tabulates prominent peaks present in the XRPD of FIG. 10(a).

FIG. 10( d) depicts an FT-IR spectrum obtained for Compound I Form L.

FIG. 10( e)) tabulates peak positions of bands present in the FT-IRspectrum of FIG. 10( d).

FIG. 10 (f) depicts Panalytical X-Pert Pro MPD PW3040 data for CompoundI Form L.

FIG. 10( g) depicts a DSC thermogram obtained for Compound I Form L.

FIG. 10( h) depicts a TGA thermogram obtained for Compound I Form L.

FIG. 10( i) depicts data for Compound I Form L.

FIG. 11 (a) depicts an XRPD for Compound I Form N.

FIG. 11( b) depicts a DSC thermogram obtained for Compound I Form N.

FIG. 11( c) depicts a TGA thermogram obtained for Compound I Form N.

FIG. 12 tabulates a single crystal structure summary for solid forms ofCompound I.

DEFINITIONS

The terms “treat”, “treating” or “treatment”, as used herein, refer to amethod of alleviating or abrogating a disease and/or its attendantsymptoms. The terms “prevent”, “preventing” or “prevention”, as usedherein, refer to a method of barring a subject from acquiring a disease.

The term “therapeutically effective amount” refers to that amount of thecompound being administered sufficient to prevent development of oralleviate to some extent one or more of the symptoms of the condition ordisorder being treated.

The term “subject” is defined herein to include animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. Inpreferred embodiments, the subject is a human.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds which are prepared with relatively nontoxic acids.Acid addition salts can be obtained by contacting the neutral form ofsuch compounds with a sufficient amount of the desired acid, either neator in a suitable inert solvent. Examples of pharmaceutically acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,citric, tartaric, methanesulfonic, and the like. Also included are saltsof amino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge, et al. (1977) J. Pharm. Sci. 66:1-19).

A pharmaceutically acceptable salt form of a compound can be prepared insitu during the final isolation and purification of the compound, orseparately by reacting the free base functionality with a suitableorganic or inorganic acid. Examples of typical pharmaceuticallyacceptable, nontoxic acid addition salts are salts of an amino groupformed with inorganic acids such as hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid, and perchloric acid, or with organicacids such as acetic acid, oxalic acid, maleic acid, tartaric acid,citric acid, succinic acid, or malonic acid or by using other methodsused in the art such as ion exchange. Other pharmaceutically acceptablesalts can include adipate, alginate, ascorbate, aspartate,benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,camphorsulfonate, citrate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,glycerophosphate, gluconate, hernisulfate, heptanoate, hexanoate,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts can include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate, and aryl sulfonate.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

The terms, “polymorphs” and “polymorphic forms” and related terms referto one of a variety of different crystal structures that can be adoptedby a particular compound. In some embodiments, polymorphs occur when aparticular chemical compound can crystallize in more than one structuralarrangement. Different polymorphs may have different physical propertiessuch as, for example, melting temperatures, heats of fusion,solubilities, dissolution rates and/or vibrational spectra as a resultof the arrangement or conformation of the molecules in the crystallattice. The differences in physical properties exhibited by polymorphsaffect pharmaceutical parameters such as storage stability,compressibility and density (important in formulation and productmanufacturing), and dissolution rates (an important factor indetermining bioavailability). Differences in stability can result fromchanges in chemical reactivity (e.g., differential oxidation, such thata dosage form discolors more rapidly when comprised of one polymorphthan when comprised of another polymorph) or mechanical changes (e.g.,tablets crumble on storage as a kinetically favored polymorph convertsto thermodynamically more stable polymorph) or both (e.g., tablets ofone polymorph are more susceptible to breakdown at high humidity). As aresult of solubility/dissolution differences, in the extreme case, somepolymorphic transitions may result in lack of potency or, at the otherextreme, toxicity. In addition, the physical properties of the crystalmay be important in processing, for example, one polymorph might be morelikely to form solvates or might be difficult to filter and wash free ofimpurities (i.e., particle shape and size distribution might bedifferent between one polymorph relative to the other).

Polymorphs of a molecule can be obtained by a number of methods, asknown in the art. Such methods include, but are not limited to, meltrecrystallization, melt cooling, solvent recrystallization, desolvation,rapid evaporation, rapid cooling, slow cooling, vapor diffusion andsublimation. Polymorphism can be detected using thermal analysis, e.g.,differential scanning calorimetry (DSC) and thermogravimetry (TGA).

Techniques for characterizing polymorphs include, but are not limitedto, differential scanning calorimetry (DSC), X-ray powder diffractometry(XRPD), single crystal X-ray diffractometry, vibrational spectroscopy,e.g, IR and Raman spectroscopy, solution calorimetry, solid state NMR,hot stage optical microscopy, scanning electron microscopy (SEM),electron crystallography and quantitative analysis, particle sizeanalysis (PSA), surface area analysis, solubility studies anddissolution studies.

The term, “solvate”, as used herein, refers to a crystal form of asubstance which contains solvent. The term “hydrate” refers to a solvatewherein the solvent is water.

The term, “desolvated solvate”, as used herein, refers to a crystal formof a substance which can only be made by removing the solvent from asolvate.

The term “prodrug”, as used herein, refers to structurally modifiedforms of the compound that readily undergo chemical changes underphysiological conditions to provide the compound. Additionally, prodrugscan be converted to the compound by chemical or biochemical methods inan ex vivo environment. Prodrugs are often useful because, in somesituations, they may be easier to administer than the compound, orparent drug. They may, for instance, be bioavailable by oraladministration whereas the parent drug is not. The prodrug may also haveimproved solubility in pharmaceutical compositions over the parent drug.A wide variety of prodrug derivatives are known in the art, such asthose that rely on hydrolytic cleavage or oxidative activation of theprodrug. An example, without limitation, of a prodrug would be acompound which is administered as an ester (the “prodrug”), but then ismetabolically hydrolyzed to the carboxylic acid, the active entity.

As used herein, the term “about”, when used in reference to any degree2-theta value recited herein, refers to the stated value±0.1 degree2-theta.

The term “anhydrous”, as used herein, refers to a form of a compoundthat is substantially free of water. One of skill in the art willappreciate that an anhydrous solid can contain various amounts ofresidual water wherein that water is not incorporated in the crystallinelattice. Such incorporation of residual water can depend upon acompound's hygroscopicity and storage conditions.

The term “hydrate”, as used herein, refers to a crystal form adopted bya particular compound in which either a stoichiometric ornon-stoichiometric amount of water is incorporated into the crystallattice.

The term “carrier”, as used herein, refers to any chemical (e.g.,solvents, diluents, or other liquid vehicles, dispersion or suspensionaids, surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants, and the like, assuited to the particular dosage form desired, Remington's PharmaceuticalSciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton,Pa., 1975)) consistent with the stability of Compound I. In certainembodiments, the term “carrier” refers to a pharmaceutically acceptablecarrier. An exemplary carrier herein is water.

The term “characterized by”, as used herein, means that a crystallineform is associated with a particular data set (e.g., one or more XRPDpeaks, melting point, DSC, TGA, DSC-TGA, and/or other characterizationmethods known to one of skill in the art, or combinations thereof). Insome embodiments, a solid form is “characterized by” a set of data whenthat set of data distinguishes the form from other known forms of therelevant compound and/or detects the presence of a particular form in acomposition containing other entities (e.g., other forms of the compoundand/or components that are not the compound). The present disclosurecontains representative data obtained from a variety of different solidforms; comparison of provided data allows one of skill in the art todetermine data sets that “characterize” any of the solid forms describedherein.

The term “electrolyte supplementation”, as used herein, refers toadministration to a subject of a composition comprising one or moreelectrolytes in order to increase serum electrolyte levels in thesubject. For purposes of the present disclosure, when electrolytesupplementation is administered “prior to, during, or after” therapy, itmay be administered prior to initiation of combination inhibitor therapy(i.e., prior to administration of any dose) or prior to, concurrentlywith, or after any particular dose or doses.

The term “formulation”, as used herein, refers to a composition thatincludes at least one active compound (e.g., at least a provided form ofCompound I) in combination with one or more excipients or otherpharmaceutical additives for administration to a patient. In general,particular excipients and/or other pharmaceutical additives are selectedin accordance with knowledge in the art to achieve a desired stability,release, distribution and/or activity of active compound(s).

The phrase “in combination”, as used herein, refers to administration oftwo or more agents to a subject. It will be appreciated that two or moreagents are considered to be administered “in combination” whenever asubject is simultaneously exposed to both (or more) of the agents. Eachof the two or more agents may be administered according to a differentschedule; it is not required that individual doses of different agentsbe administered at the same time, or in the same composition. Rather, solong as both (or more) agents remain in the subject's body, they areconsidered to be administered “in combination”.

The term “isostructural” or “isostructure”, as used herein, refers totwo or more solid forms of a compound containing essentially the samethree-dimensional arrangement of geometrically similar structural units.In some embodiments, “isostructural” forms show with similar oridentical unit cell dimensions, the same space group, and similar oridentical atomic coordinates for common atoms. In some embodiments,“isostructural” forms have the same structure, but not the same celldimensions nor the same chemical composition, and have comparablevariability in their atomic coordinates to that of the cell dimensionsand chemical composition. In some embodiments, the present disclosuredescribes a set of isostructural forms of Compound I including, forexample, taken from forms of Compound I described infra. In someembodiments, the present disclosure describes a set of isostructuralforms including, for example, Form J and/or Form D. In some embodiments,the present disclosure describes a set of isostructural forms including,for example, Form E and/or Form H. In some embodiments, the presentdisclosure describes a set of isostructural forms including, forexample, Form C and/or the methanol solvate reported in Shigematsu etal., The Journal of Antibiotics, Vol. 47, No. 3, “FR901228, A NovelAntitumor Bicyclic Depsipeptide Produced by Chromobacterium violaceumNo. 968, pp. 311-314 (March 1994).

The term “lyophilize”, as used herein, refers to the process ofisolating a solid substance from solution and/or removal of solvent. Insome embodiments, this may be achieved by various techniques known toone of skill in the art, including, for example, evaporation (e.g.,under vacuum, for example by rotary evaporation), freeze drying, and/orfreezing the solution and vaporizing frozen solvent away under vacuumconditions, etc.

The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques.

The term “substantially all”, as used herein, when used to describeX-ray powder diffraction (“XRPD”) peaks of a compound typically meansthat the XRPD of that compound includes at least about 80% of the peakswhen compared to a reference. For example, when an XRPD is said toinclude “substantially all” of the peaks in a reference list, or all ofthe peaks in a reference XRPD, it means that the XRPD includes at least80% of the peaks in the specified reference. In other embodiments, thephrase “substantially all” means that the XRPD of that compound includesat least about 85, 90, 95, 97, 98, or 99% of the peaks when compared toa reference.

The term “substantially free of”, as used herein, means containing nomore than an insignificant amount. In some embodiments, a composition orpreparation is “substantially free of” a recited element if it containsless than 5%, 4%, 3%, 2%, or 1%, by weight of the element. In someembodiments, the composition or preparation contains less than 0.9%,0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less of the recitedelement. In some embodiments, the composition or preparation contains anundetectable amount of the recited element.

The term “substantially similar,” as used herein, refers to data sets(e.g., spectra/thermograms) that share similarities with each otherand/or that differentiate them from one or more reference data sets. Incertain embodiments, data sets are considered to be “substantiallysimilar” to one another when their similarities to each other anddifferences from one or more reference data sets are sufficient topermit a conclusion that the two compared data sets are taken of thesame form of a compound, whereas the reference data set is/are taken ofa different form of the compound. In some embodiments, two“substantially similar” data sets are the same (i.e., are identicalwithin experimental error). In some embodiments, presence in a data setof one or more data points characteristic of a particular form of acompound, but absence of some or all data points that are characteristicof a different form (e.g., data points that are usually present inreference data set) defines data sets as substantially similar to eachother.

The expression “unit dose”, as used herein, refers to a physicallydiscrete unit of a formulation appropriate for a subject to be treated(e.g., for a single dose); each unit containing a predetermined quantityof an active agent selected to produce a desired therapeutic effect (itbeing understood that multiple doses may be required to achieve adesired or optimum effect), optionally together with a pharmaceuticallyacceptable carrier, which may be provided in a predetermined amount. Theunit dose may be, for example, a volume of liquid (e.g., an acceptablecarrier) containing a predetermined quantity of one or more therapeuticagents, a predetermined amount of one or more therapeutic agents insolid form, a sustained release formulation or drug delivery devicecontaining a predetermined amount of one or more therapeutic agents,etc. It will be appreciated that a unit dose may contain a variety ofcomponents in addition to the therapeutic agent(s). For example,acceptable carriers (e.g., pharmaceutically acceptable carriers),diluents, stabilizers, buffers, preservatives, etc., may be included asdescribed infra. It will be understood, however, that the total dailyusage of a formulation of the present disclosure will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular subject or organism maydepend upon a variety of factors including the disorder being treatedand the severity of the disorder; activity of specific active compoundemployed; specific composition employed; age, body weight, generalhealth, sex and diet of the subject; time of administration, and rate ofexcretion of the specific active compound employed; duration of thetreatment; drugs and/or additional therapies used in combination orcoincidental with specific compound(s) employed, and like factors wellknown in the medical arts.

DETAILED DESCRIPTION

It has been found that Compound I can exist in a variety of solid forms.Such solid forms include neat crystal forms. Such solid forms alsoinclude solvated forms and amorphous forms. The present disclosureprovides certain such solid forms of Compound I. In certain embodiments,the present disclosure provides compositions comprising Compound I in aform described herein. In some embodiments of provided compositions,Compound I is present as a mixture of one or more solid forms; in someembodiments of provided compositions, Compound I is present in only asingle form.

In certain embodiments of the present disclosure, Compound I is providedas a crystalline solid. In certain embodiments, Compound I is providedas a crystalline solid substantially free of amorphous Compound I. Incertain embodiments, Compound I is provided as an amorphous form. Incertain embodiments, Compound I is provided as a solvated form.

In some embodiments, all of Compound I that is present in a particularcomposition is present in a particular form; in some such embodiments,the composition is substantially free of any other form of Compound I.In some embodiments, a composition comprises a Compound I, present in acombination of different forms.

In some embodiments, the present disclosure provides a lyophilate ofCompound I containing one or more solid forms described herein. In someembodiments, a lyophilate comprises amorphous Compound I. In someembodiments, a lyophilate comprises one or more crystalline forms. Insome embodiments, a lyophilate is substantially free of one or morecrystalline forms. In some embodiments, a lyophilate is substantiallyfree of any crystalline form.

In some embodiments, the present disclosure provides one or more solidforms as described herein, in combination with one or more othercomponents. In some such embodiments, other components are selected fromthe group consisting of, for example, buffers, carriers, crystallizationinhibitors, diluents, excipients, pH adjustors, solvents, or otherpharmaceutical additives for administration to a patient.

In certain embodiments, where Compound I is in amorphous form (e.g., incertain lyophilates), such compositions comprise one or morecrystallization inhibitors.

To characterize individual crystal forms of a particular compound,and/or to detect the presence of a particular form in a complexcomposition techniques known to those of skill in the art, such as thatX-ray diffraction patterns, differential scanning calorimeterthermograms, thermal gravimetic analyzer thermograms, melting pointinformation, polarized light microscopy, hotstage microscopyphotomicrographs, dynamic vapor sorption/desorption information, watercontent, IR spectra, NMR spectra, and hygroscopicity profiles, to name afew, are used. Those of skill in the art will further appreciate thatprecise identity of all peaks, for example, in an X-ray diffractionpattern, is not required to reveal a match of crystal form. Rather,presence or absence of particular characteristic peaks, and/or patternsof peaks and intensities, are typically both necessary and sufficient tocharacterize and/or identify a particular form.

Solid Forms

The present disclosure provides solid forms of Compound I. In certainembodiments, the present disclosure provides Compound I in a crystallineform. In some embodiments, crystalline forms are substantially free ofsolvent. In some embodiments, crystalline forms are a solvate. In someembodiments, the present disclosure provides Compound I in an amorphousform. A summary table of the romidepsin solid forms (Table 1 is providedbelow.

In one embodiment, solid forms of Compound I provided herein possessimproved properties. These properties include, but are not limited to,bioavailability, (including, without limitation, light and heatstability), solubility, compressability, flowability, electrostaticproperties, bulk density, and rate of dissolution.

TABLE 1 Romidepsin Solid Forms Solid Form Form A (Desired Form) Form BForm C Form D Form E Form H Crystallization Acetone/waterAcetone/hexanes Acetone/water Acetone/hexanes Tert-butyl Chloroformsolvent system (85/15)¹ (85/15) or acetone (1/3) (1/4) or acetonealcohol/water (60/40) Crystallization Room Room Cold (−5° C.) Cold (−20°C.) Room rotary temperature temperature temperature temperatureevaporation at 60° C. Thermal analysis 254.4° C. (endo) ⁴ 258.3° C.(endo) ⁴  83.6° C. (endo)  91.4° C. (exo) 158.1° C. (endo)  96.3° C.(endo) (DSC) ³ 126.8° C. (endo) 260.6° C. (endo) 255.2° C. (endo) 256.6°C. (endo) 171.9° C. (exo) 257.8° C. (endo) Slurry Remains A A + B → AA + C → A A + D → A, trace C A + E → A, trace E — interconversion (2hrs) (2 hrs) (2.5 hrs) (2.5 hrs) ¹Crystallization occurs with additionof water to a final 15/85 acetone/water ratio. ²Crystallization occurswith seeding after addition of water to a final 1/3 acetone/water ratio.³ Samples analyzed in a crimped aluminum pan at 10° C./min, unless notedotherwise ⁴ Sample analyzed in a crimped aluminum pan at 10° C./min withmanual pinhole. Solid Form Form F Form I Form J Form K Form L Form NCrystallization Chloroform Chloroform slurry Methyl ethyl NitromethaneDissolved solids in Nitromethane solvent system or vapor stress ketoneacetone and diffused with methanol Crystallization Room Room Room RoomRoom Room temperature temperature temperature temperature temperaturetemperature temperature or cold (−20° C.) Thermal analysis  83.6° C.(minor endo)  73.8° C. (endo) 130.3° C. (endo)  68.8° C. (endo) 168.2°C. (endo) 150.0° C. (event) (DSC)¹  97.3° C. (endo) 100.2° C. (endo)260.0° C. (endo)²  81.3° C. (endo) 259.2° C. (endo) 259.1° C. (endo)256.4° C. (endo) 257.8° C. (endo) 145.9° C. (endo) 257.2° C. (endo)Slurry — — — — — — interconversion ¹Samples analyzed in a crimpedaluminum pan at 10° C./min, unless noted otherwise. ²Sample analyzed ina hermetically-sealed aluminum pan at 10° C./min.

Crystalline Form A and Crystalline Form B

Compound I is known to exist in different crystalline forms, known asForm A and Form B. These forms are described in PCT Publication No.WO02/020817, filed Aug. 22, 2001, which is incorporated herein byreference.

Crystalline Form C

In some embodiments, the present disclosure provides Form C of CompoundI, and compositions comprising Form C. In some embodiments, acomposition comprising Compound I, contains at least some of Compound Iin a crystalline form, which crystalline form comprises Form C.

In one embodiment, Compound I Form C is obtained from an acetone/watermixture.

In one embodiment, Compound I Form C is analyzed by one or more ofoptical microscropy, X-ray powder diffraction, differential scanningcalorimetry, modulated differential scanning calorimetry,thermogravimetric analysis, infrared spectroscopy, nuclear magneticresonance spectroscopy, and raman spectroscopy.

In some embodiments, crystalline Form C of Compound I is characterizedby the presence of one or more, two or more, three or more, four ormore, five or more, or six or more peaks from its XRPD pattern, whichpeaks, when taken alone or together with other characteristic data,distinguish Form C from other forms, as described infra. In oneembodiment, Compound I Form C shows an X-ray diffraction having peakssubstantially similar to those in FIG. 1( c). For example, Form C ischaracterized by a peak in the XRPD at about 11.45 2θ. Othercharacteristic peaks include 8.28, 12.19, and 21.13 2θ.

As described herein, crystalline Form C of Compound I is characterized,for example, by some or all, of the exemplary data provided in FIGS. 1(c) through 1(q), infra (and discussed in Example 2). In one embodiment,a DSC thermogram obtained for Compound I Form C exhibits broadendothermic events at ˜140° C. (min); an endotherm at ˜257° C. (min);and a minor exothermic event at approximately 177° C. (max). In oneembodiment, a TGA thermogram obtained for Compound I Form C exhibits aweight loss of ˜5.3%.

In some embodiments, Form C is isostructural with the methanol solvatereported in Shigematsu et al., The Journal of Antibiotics, Vol. 47(3)“FR901228, A Novel Antitumor Bicyclic Depsipeptide Produced byChromobacterium violaceum No. 968, pp. 311-314 (March 1994).

Crystalline Form D

In some embodiments, the present disclosure provides a crystalline formobtained from acetone. In some embodiments, the acetone is cold. In someembodiments, the acetone has a temperature of −15° C. or lower (e.g.,−25° C., −35° C., −50° C., −70° C. or lower). In some embodiments, sucha crystalline form is a solvate. In some embodiments, an acetone solvateis referred to as Form D of Compound I. In some embodiments, Form D maybe isostructural with Form J described infra.

In one embodiment, Compound I Form D is analyzed by one or more ofoptical microscopy, X-ray powder diffraction, differential scanningcalorimetry, modulated differential scanning calorimetry,thermogravimetric analysis, infrared spectroscopy, nuclear magneticresonance spectroscopy, and raman spectroscopy.

In some embodiments, the present disclosure provides Form D of CompoundI, and compositions comprising Form D. In some embodiments, acomposition comprising Compound I contains at least some of Compound Iin a crystalline form, which crystalline form comprises Form D. In someembodiments, a composition comprising Compound I contains at least someof Compound I in a solvated crystalline form, which crystalline formcomprises Form D. In certain embodiments, the solvated form is anacetone solvate.

In some embodiments, crystalline Form D of Compound I is characterizedby the presence of one or more, two or more, three or more, four ormore, five or more, or six or more peaks from its XRPD pattern, whichpeaks, when taken alone or together with other characteristic data,distinguish Form D from other forms, as described infra. In oneembodiment, Compound I Form D shows an X-ray diffraction having peakssubstantially similar to those in FIG. 2( a). For example, Form D ischaracterized by a peak in the XRPD at about 7.54 2θ. Othercharacteristic peaks include 11.86 and 16.66 2θ.

As described herein, Compound I Form D is characterized by some or allof the exemplary data provided in 2(a) through 2(f), infra (anddiscussed in Example 3). In one embodiment, a DSC thermogram obtainedfor Compound I Form D exhibits a small endothermic event at ˜91° C.(min); and an endotherm at ˜261° C. (min); followed by apparentdecomposition. In one embodiment, a TGA thermogram obtained for CompoundI Form D exhibits a weight loss of ˜10.9%.

Crystalline Form E

In some embodiments, the present disclosure provides a crystalline formobtained from t-butanol. In some embodiments, the present disclosureprovides a crystalline form obtained from a mixture of t-butanol andwater. In some embodiments, such a crystalline form is a solvate. Insome embodiments, a t-butanol solvate is referred to as Form E ofCompound I. In some embodiments, Form E may be isostructural with Form Hdescribed infra.

In some embodiments, the present disclosure provides Form E of CompoundI, and compositions comprising Form E. In some embodiments, acomposition comprising Compound I, contains at least some of Compound Iin a crystalline form, which crystalline form comprises Form E. In someembodiments, a composition comprising Compound I, contains at least someof Compound I in a solvated crystalline form, which crystalline formcomprises Form E. In certain embodiments, the solvated form is at-butanol solvate.

In one embodiment, Compound I Form E is analyzed by one or more ofoptical microscopy, X-ray powder diffraction, differential scanningcalorimetry, modulated differential scanning calorimetry,thermogravimetric analysis, infrared spectroscopy, nuclear magneticresonance spectroscopy, and raman spectroscopy.

In some embodiments, crystalline Form E of Compound I is characterizedby the presence of one or more, two or more, three or more, four ormore, five or more, or six or more peaks from its XRPD pattern, whichpeaks, when taken alone or together with other characteristic data,distinguish Form E from other forms, as described infra. In oneembodiment, Compound I Form E shows an X-ray diffraction having peakssubstantially similar to those in FIG. 3( a). For example, Form E ischaracterized by a peak in the XRPD at about 10.3 2θ. Othercharacteristic peaks include 9.0, 11.7, and 20.04 2θ.

As described herein, Compound I Form E is characterized by some or allof the exemplary data provided in FIGS. 3( a) through 3(p), infra (anddiscussed in Example 4). In one embodiment, a DSC thermogram obtainedfor Compound I Form E exhibits an endothermic event at ˜158° C. (min);an endotherm at ˜255° C. (min); followed by apparent decomposition. Inone embodiment, a TGA thermogram obtained for Compound I Form E exhibitsa weight loss of ˜10.9%.

Crystalline Form F

In some embodiments, the present disclosure provides a crystalline formobtained from chloroform.

In some embodiments, the present disclosure provides Form F of CompoundI, and compositions comprising Form F. In some embodiments, acomposition comprising Compound I, contains at least some of Compound Iin a crystalline form, which crystalline form comprises Form F. In someembodiments, such a crystalline form is a solvate.

In some embodiments, Compound I Form F is analyzed by one or more ofoptical microscopy, X-ray powder diffraction, differential scanningcalorimetry, modulated differential scanning calorimetry,thermogravimetric analysis, infrared spectroscopy, nuclear magneticresonance spectroscopy, and raman spectroscopy.

In some embodiments, crystalline Form F of Compound I is characterizedby the presence of one or more, two or more, three or more, four ormore, five or more, or six or more peaks from its XRPD pattern, whichpeaks, when taken alone or together with other characteristic data,distinguish Form F from other forms, as described infra. In oneembodiment, Compound I Form F shows an X-ray diffraction having peakssubstantially similar to those in FIG. 9( a) or 9(d). For example, FormF is characterized by a peak in the XRPD at about 20.28 2θ. Othercharacteristic peaks include 10.17, 17.8, 19.34, 20.04, and 22.63 2θ.

As described herein, Compound I Form F is characterized by some or allof the exemplary data provided in 9(a) through (9 l), infra (anddiscussed in Example 5). In one embodiment, a DSC thermogram obtainedfor Compound I Form F exhibits a broad endothermic event at ˜97° C.(min); and an endotherm at ˜256° C. (min). In one embodiment, a TGAthermogram obtained for Compound I Form F exhibits a weight loss of˜17%. In one embodiment, provided is the Panalytical X-Pert Pro MPDPW3040 data for Compound I Form F obtained under the followingconditions: X-ray Tube: Cu (1.54059 A°), Voltage: 45 kV; Amperage 40 mA;Scan range: 1.00-39.98 °2θ; step size: 0.017 °2θ; collection time: 721sec.; scan speed: 3.2°/min; slit: DS:1/2°; SS: null; revolution time:1.0 sec., mode: transmission. In one embodiment, provided is the datafor Compound I Form F obtained under the following conditions: detector:DTGS KBr; number of scans: 512; resolution: 2 cm⁻¹.

Crystalline Form H

In some embodiments, the present disclosure provides a crystalline formobtained from chloroform. In some embodiments, such a crystalline formis a solvate. In some embodiments, a chloroform solvate is referred toas Form H of Compound I. In some embodiments, Form H may beisostructural with Form E described infra.

In some embodiments, the present disclosure provides Form H of CompoundI, and compositions comprising Form H. In some embodiments, acomposition comprising Compound I, contains at least some of Compound Iin a crystalline form, which crystalline form comprises Form H. In someembodiments, a composition comprising Compound I, contains at least someof Compound I in a solvated crystalline form, which crystalline formcomprises Form H. In some embodiments, the solvated form is a chloroformsolvate.

In some embodiment, Compound I Form H is analyzed by one or more ofoptical microscopy, X-ray powder diffraction, differential scanningcalorimetry, modulated differential scanning calorimetry,thermogravimetric analysis, infrared spectroscopy, nuclear magneticresonance spectroscopy, and raman spectroscopy.

In some embodiments, crystalline Form H of Compound I is characterizedby the presence of one or more, two or more, three or more, four ormore, five or more, or six or more peaks from its XRPD pattern, whichpeaks, when taken alone or together with other characteristic data,distinguish Form H from other forms, as described infra. In oneembodiment, Compound I Form H shows an X-ray diffraction having peakssubstantially similar to those in FIG. 4( a). For example, Form H ischaracterized by a peak in the XRPD at about 10.67 2θ. Othercharacteristic peaks include 8.94, 9.69, 10.51, 13.13, and 19.43 2θ.

As described herein, Compound I Form H is characterized by some or allof the exemplary data provided in 4(a) through 4(f), infra (anddiscussed in Example 6). In one embodiment, a DSC thermogram obtainedfor Compound I Form H exhibits an endothermic event at ˜96° C. (min);and an endotherm at ˜257° C. (min). In one embodiment, a TGA thermogramobtained for Compound I Form H exhibits a weight loss of ˜10.1%.

Crystalline Form I

In some embodiments, the present disclosure provides a crystalline formobtained from chloroform. In some embodiments, such a crystalline formis a solvate. In some embodiments, a chloroform solvate is referred toas Form I of Compound I.

In some embodiments, the present disclosure provides Form I of CompoundI, and compositions comprising Form I. In some embodiments, acomposition comprising Compound I, contains at least some of Compound Iin a crystalline form, which crystalline form comprises Form I. In someembodiments, a composition comprising Compound I, contains at least someof Compound I in a solvated crystalline form, which crystalline formcomprises Form I. In some embodiments, the solvated form is a chloroformsolvate.

In some embodiments, Compound I Form I is analyzed by one or more ofoptical microscopy, X-ray powder diffraction, differential scanningcalorimetry, modulated differential scanning calorimetry,thermogravimetric analysis, infrared spectroscopy, nuclear magneticresonance spectroscopy, and raman spectroscopy.

In some embodiments, crystalline Form I of Compound I is characterizedby the presence of one or more, two or more, three or more, four ormore, five or more, or six or more peaks from its XRPD pattern, whichpeaks, when taken alone or together with other characteristic data,distinguish Form I from other forms, as described infra. In oneembodiment, Compound I Form I shows an X-ray diffraction having peakssubstantially similar to those in FIG. 5( a) or 5(p). For example, FormI is characterized by a peak in the XRPD at about 20.96 2θ. Othercharacteristic peaks include 10.63, 17.97, 18.74, 19.12, and 23.18 2θ.

As described herein, crystalline Form I of Compound I is characterizedby some or all of the exemplary data provided in 5(a) through 5(y),infra (and discussed in Example 7). In one embodiment, a DSC thermogramobtained for Compound I Form I exhibits a broad endothermic event at˜74° C. (min); an endothermic event at ˜100° C. (min); and an endothermat 256.4° C. (min) (10° C./min, C). In another embodiment, a DSCthermogram obtained for Compound I Form I exhibits a broad endothermicevent at ˜88° C. (min); an endothermic event at ˜113° C. (min); and anendotherm at ˜256° C. (min) (10° C./min, C). In one embodiment, a TGAthermogram obtained for Compound I Form I exhibits a weight loss of˜33%. In another embodiment, a TGA thermogram obtained for Compound IForm I exhibits a weight loss of ˜27%. In one embodiment, provided isthe Panalytical X-Pert Pro MPD PW3040 data for Compound I Form Iobtained under the following conditions: X-ray Tube: Cu (1.54059 A°),Voltage: 45 kV; Amperage 40 mA; Scan range: 1.00-39.99 °2θ; step size:0.017 °2θ; collection time: 718 sec.; scan speed: 3.3°/min; slit:DS:1/2°; SS: null; revolution time: 1.0 sec., mode: transmission. In oneembodiment, provided is the data for Compound I Form I obtained underthe following conditions: detector: DTGS KBr; number of scans: 512;resolution: 2 cm⁻¹.

Crystalline Form J

In some embodiments, the present disclosure provides a crystalline formobtained from methylethylketone. In some embodiments, such a crystallineform is a solvate. In some embodiments, a methylethylketone solvate isreferred to as Form J of Compound I. In some embodiments, Form J may beisostructural with Form D described infra.

In some embodiments, the present disclosure provides Form J of CompoundI, and compositions comprising Form J of Compound I. In someembodiments, a composition comprising Compound I, contains at least someof Compound I in a crystalline form, which crystalline form comprisesForm J. In some embodiments, a composition comprising Compound I,contains at least some of Compound I in a solvated crystalline form,which crystalline form comprises Form J. In certain embodiments, thesolvated form is a methylethylketone solvate.

In some embodiments, Compound I Form J is analyzed by one or more ofoptical microscopy, X-ray powder diffraction, differential scanningcalorimetry, modulated differential scanning calorimetry,thermogravimetric analysis, infrared spectroscopy, nuclear magneticresonance spectroscopy, and raman spectroscopy.

In some embodiments, crystalline Form J of Compound I is characterizedby the presence of one or more, two or more, three or more, four ormore, five or more, or six or more peaks from its XRPD pattern, whichpeaks, when taken alone or together with other characteristic data,distinguish Form J from other forms, as described infra. In oneembodiment, Compound I Form J shows an X-ray diffraction having peakssubstantially similar to those in FIG. 6( k). For example, Form J ischaracterized by a peak in the XRPD at about 15.24 2θ. Othercharacteristic peaks include 7.44, 11.80, and 16.60 2θ.

As described herein, crystalline Compound I Form J is characterized bysome or all of the exemplary data provided in 6(a) through 6(u), infra(and discussed in Example 8). In one embodiment, a DSC thermogramobtained for Compound I Form J exhibits a broad endothermic event at˜130° C. (min); and an endotherm at ˜260° C. (min). In one embodiment, aTGA thermogram obtained for Compound I Form J exhibits a weight loss of˜12%. In one embodiment, provided is the Panalytical X-Pert Pro MPDPW3040 data for Compound I Form J obtained under the followingconditions: X-ray Tube: Cu (1.54059 A°), Voltage: 45 kV; Amperage 40 mA;Scan range: 1.00-39.99 °2θ; step size: 0.017 °2θ; collection time: 718sec.; scan speed: 3.3°/min; slit: DS:1/2°; SS: null; revolution time:1.0 sec., mode: transmission. In one embodiment, provided is the datafor Compound I Form J obtained under the following conditions: detector:DTGS KBr; number of scans: 512; resolution: 2 cm⁻¹.

Crystalline Form K

In some embodiments, the present disclosure provides Form K of CompoundI, and compositions comprising Form K. In some embodiments, acomposition comprising Compound I contains at least some of Compound Iin a crystalline form, which crystalline form comprises Form K. In someembodiments, a composition comprising Compound I contains at least someof Compound I in a solvated crystalline form, which crystalline formcomprises Form K. In one embodiment, Compound I Form K is obtained fromnitromethane. In one embodiment, Compound I Form K is a nitromethanesolvate.

In some embodiments, Compound I Form K is analyzed by one or more ofoptical microscopy, X-ray powder diffraction, differential scanningcalorimetry, modulated differential scanning calorimetry,thermogravimetric analysis, infrared spectroscopy, nuclear magneticresonance spectroscopy, and raman spectroscopy.

In some embodiments, crystalline Form K of Compound I is characterizedby the presence of one or more, two or more, three or more, four ormore, five or more, or six or more peaks from its XRPD pattern, whichpeaks, when taken alone or together with other characteristic data,distinguish Form K from other forms, as described infra. In oneembodiment, Compound I Form K shows an X-ray diffraction having peakssubstantially similar to those in FIG. 8( c). For example, Form K ischaracterized by a peak in the XRPD at about 7.89 2θ. Othercharacteristic peaks include 11.25, 16.81, 19.40, and 20.96 2θ.

As described herein, Compound I Form K is characterized by some or allof the exemplary data provided in 8(a) through 8(l), infra (anddiscussed in Example 10). In one embodiment, a DSC thermogram obtainedfor Compound I Form K exhibits a broad endothermic event at ˜62° C.(min); another broad endothermic event at ˜155° C. (min); and anendotherm at ˜257° C. (min). In another embodiment, a DSC thermogramobtained for Compound I Form K exhibits a broad endothermic event at˜69° C. and 81° C.; another broad endothermic event at ˜146° C. (min);and an endotherm at ˜257° C. (min). In one embodiment, a TGA thermogramobtained for Compound I Form K exhibits a weight loss of ˜9.5%. In oneembodiment, provided is the Panalytical X-Pert Pro MPD PW3040 data forCompound I Form K obtained under the following conditions: X-ray Tube:Cu (1.54059 A°), Voltage: 45 kV; Amperage 40 mA; Scan range: 1.00-39.99°2θ; step size: 0.017 °2θ; collection time: 717 sec.; scan speed:3.3°/min; slit: DS:1/2°; SS: null; revolution time: 1.0 sec., mode:transmission. In one embodiment, provided is the data for Compound IForm K obtained under the following conditions: detector: DTGS KBr;number of scans: 512; resolution: 2 cm⁻¹.

Crystalline Form L

In some embodiments, the present disclosure provides a crystalline formobtained from acetone and diffused with methanol.

In some embodiments, the present disclosure provides Form L of CompoundI, and compositions comprising Form L. In some embodiments, acomposition comprising Compound I, contains at least some of Compound Iin a crystalline form, which crystalline form comprises Form L. In oneembodiment, Compound I Form L is a methanol solvate.

In some embodiments, Compound I Form L is analyzed by one or more ofoptical microscopy, X-ray powder diffraction, differential scanningcalorimetry, modulated differential scanning calorimetry,thermogravimetric analysis, infrared spectroscopy, nuclear magneticresonance spectroscopy, and raman spectroscopy.

In some embodiments, crystalline Form L of Compound I is characterizedby the presence of one or more, two or more, three or more, four ormore, five or more, or six or more peaks from its XRPD pattern, whichpeaks, when taken alone or together with other characteristic data,distinguish Form L from other forms, as described infra. In oneembodiment, Compound I Form L shows an X-ray diffraction having peakssubstantially similar to those in FIG. 10( a). For example, Form L ischaracterized by a peak in the XRPD at about 21.46 2θ. Othercharacteristic peaks include 8.26, 10.05, 11.59, and 12.31 2θ.

As described herein, Compound I Form L is characterized by some or allof the exemplary data provided in 10(a) through 10(i), infra (anddiscussed in Example 11). In one embodiment, a DSC thermogram obtainedfor Compound I Form L exhibits an endothermic event at ˜168° C. (min);and an endotherm at ˜259° C. (min). In one embodiment, a TGA thermogramobtained for Compound I Form L exhibits a weight loss of ˜6%. In oneembodiment, provided is the Panalytical X-Pert Pro MPD PW3040 data forCompound I Form L obtained under the following conditions: X-ray Tube:Cu (1.54059 A°), Voltage: 45 kV; Amperage 40 mA; Scan range: 1.00-39.98°2θ; step size: 0.017 °2θ; collection time: 716 sec.; scan speed:3.2°/min; slit: DS:1/2°; SS: null; revolution time: 1.0 sec., mode:transmission. In one embodiment, provided is the data for Compound IForm L obtained under the following conditions: detector: DTGS KBr;number of scans: 512; resolution: 2 cm⁻¹.

Crystalline Form N

In some embodiments, the present disclosure provides a crystalline formobtained from nitromethane.

In some embodiments, the present disclosure provides Form N of CompoundI, and compositions comprising Form N. In some embodiments, acomposition comprising Compound I, contains at least some of Compound Iin a crystalline form, which crystalline form comprises Form N. In oneembodiment, Form N of Compound I is a nitromethane solvate.

In some embodiments, Compound I Form N is analyzed by one or more ofoptical microscopy, X-ray powder diffraction, differential scanningcalorimetry, modulated differential scanning calorimetry,thermogravimetric analysis, infrared spectroscopy, nuclear magneticresonance spectroscopy, and raman spectroscopy.

In some embodiments, crystalline Form N of Compound I is characterizedby the presence of one or more, two or more, three or more, four ormore, five or more, or six or more peaks from its XRPD pattern, whichpeaks, when taken alone or together with other characteristic data,distinguish Form N from other forms, as described infra. In oneembodiment, Compound I Form N shows an X-ray diffraction having peakssubstantially similar to those in FIG. 11( a). For example, Form N ischaracterized by a peak in the XRPD at about 8.92 2θ. Othercharacteristic peaks include 7.07, 9.76, 10.75, 11.22, 15.46, 20.37, and21.31 2θ.

As described herein, Compound I Form N is characterized by some or allof the exemplary data provided in 11(a) through 11(c), infra (anddiscussed in Example 12). In one embodiment, a DSC thermogram obtainedfor Compound I Form N exhibits an endotherm at ˜150° C. (min). In oneembodiment, a TGA thermogram obtained for Compound I Form N exhibits aweight loss of ˜5%. In one embodiment, provided is the PanalyticalX-Pert Pro MPD PW3040 data for Compound I Form N obtained under thefollowing conditions: X-ray Tube: Cu (1.54059 A°), Voltage: 45 kV;Amperage 40 mA; Scan range: 1.00-39.99 °2θ; step size: 0.017 °2θ;collection time: 717 sec.; scan speed: 3.3°/min; slit: DS:1/2°; SS:null; revolution time: 1.0 sec., mode: transmission.

Amorphous Form

In some embodiments, the present disclosure provides amorphous CompoundI, and compositions comprising amorphous Compound I. In someembodiments, the present disclosure provides compositions comprisingCompound I in which substantially all of Compound I is an amorphous form(i.e., the composition is substantially free of crystalline compound I).In some embodiments, the present disclosure provides compositionscontaining Compound I in which at least some of the Compound I is in aform other than amorphous (e.g., is in a crystalline form such as, forexample, Form A, Form B, Form C, Form D, Form E, Form F, Form H, Form I,Form J, Form K, Form L, Form N, and combinations thereof).

In some embodiments, amorphous Compound I is characterized by theabsence of defined peaks above background in an XRPD pattern. In someembodiments, amorphous Compound I is characterized by the absence ofcharacteristic peaks that may be present in Compound I Form A, Form B,Form C, Form D, Form E, Form F, Form H, Form I, Form J, Form K, Form L,Form N, and combinations thereof. In some embodiments, amorphousCompound I is characterized by having a powder X-ray diffraction patternsubstantially similar to FIG. 7( a). In some embodiments, amorphousCompound I is obtained from a water/dichloromethane mixture, or anisopropanol-trifluoroethanol/methanol mixture

As described herein, amorphous Compound I is characterized by theexemplary data provided in 7(a) through 7(f), infra (see Example 9). Inone embodiment, a DSC thermogram obtained for amorphous Compound Iexhibits a glass transition temperature of ˜91° C. In one embodiment, aTGA thermogram obtained for amorphous Compound I exhibits a weight lossof ˜3.5%.

Compositions Comprising Provided Forms of Compound I

The present disclosure provides compositions that comprise and/or areprepared from solid forms of Compound I as described herein. Any of theforms provided herein of Compound I may be incorporated into acomposition. In some embodiments, the present disclosure providespharmaceutical compositions that comprise and/or are prepared from solidforms of Compound I as described herein. In some embodiments, apharmaceutical composition comprises a therapeutically effective amountof Compound I and at least one pharmaceutically acceptable carrier orexcipient.

In some embodiments, compositions comprising Compound I are provided aslyophilates. In some embodiments, the present disclosure provides alyophilate of Compound I comprising one or more solid forms describedherein. In some embodiments, a lyophilate comprises amorphous CompoundI. In some embodiments, a lyophilate comprises one or more crystallineforms. In some embodiments, a lyophilate is substantially free of one ormore crystalline forms. In some embodiments, a lyophilate issubstantially free of any crystalline form.

In some embodiments, the present disclosure provides compositionscomprising or prepared from Compound I solid forms described herein,which compositions further comprise one or more additional components.

In some embodiments, provided compositions comprise, in addition toCompound I, at least one other component, such as a carrier (e.g.,pharmaceutically acceptable carrier). Except insofar as any conventionalcarrier medium is incompatible with compounds or forms described herein,such as by producing any undesirable biological effect or otherwiseinteracting in a deleterious manner with any other component(s) ofcompositions and/or the use thereof is contemplated to be within thescope of this disclosure.

In some embodiments, materials which can serve as acceptable carriers(e.g., pharmaceutically acceptable carriers) include, but are notlimited to, sugars such as lactose, glucose, and sucrose; starches suchas corn starch and potato starch; cellulose and its derivatives such assodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate;powdered tragacanth; malt; gelatin; talc; Cremophor; Solutol; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil; sunflower oil; sesame oil; olive oil; corn oil andsoybean oil; glycols such a propylene glycol; esters such as ethyloleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions; as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate; as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator.

Compositions comprising Compound I as described herein may be formulatedorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. In some embodiments,compositions are administered orally or parenterally.

In some embodiments, compositions are administered parenterally. In someembodiments, compositions are administered intraperitoneally orintravenously.

As is known in the art, injectable formulations are often provided assolutions or suspensions, e.g., aqueous or oleaginous suspension. Suchsolutions or suspensions may be formulated according to techniques knownin the art, for example, using suitable dispersing or wetting agents andsuspending agents. Injectable formulations are typically sterile. Insome embodiments, an injectable solution or suspension comprises anon-toxic parenterally acceptable diluent or solvent. Exemplary vehiclesand solvents typically employed include water, Ringer's solution,isotonic sodium chloride solution, acetone, chloroform, dichloromethane,isopropanol, methanol, methylethylketone, tert-butyl alcohol,trifluoroethanol and 1,3-butanediol, and combinations thereof.

In some embodiments, sterile, fixed oils are conventionally employed asa solvent or suspending medium. Any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. Fatty acids, such as oleicacid and its glyceride derivatives are often useful in the preparationof injectables, as are natural pharmaceutically-acceptable oils, such asolive oil or castor oil, including their polyoxyethylated versions. Insome embodiments, such oil solutions or suspensions contain a long-chainalcohol diluent or dispersant, such as carboxymethyl cellulose orsimilar dispersing agents that are commonly used in the formulation ofpharmaceutically acceptable dosage forms including emulsions andsuspensions. In some embodiments, commonly used surfactants, such asTweens, Spans and other emulsifying agents or bioavailability enhancerswhich are commonly used in the manufacture of acceptable (e.g.,pharmaceutically acceptable) solid, liquid, or other dosage forms mayalso be used for the purposes of formulation.

Orally acceptable dosage forms include, but are not limited to,capsules, tablets, aqueous suspensions or solutions. In the case oftablets for oral use, carriers commonly used include lactose and cornstarch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents commonly include lactose and dried cornstarch. When aqueoussuspensions are prepared for oral delivery, the active ingredient istypically combined with emulsifying and suspending agents, optionallymuch as discussed above with respect to parenteral formulations. Ifdesired, certain sweetening, flavoring or coloring agents may also beadded.

Administration of oral compositions can be desirably linked to periodsof food intake. For example, in some embodiments, oral compositions areadministered with food; in some embodiments, oral compositions areadministered without food, or within a particular time frame relative toconsumption of food. In some embodiments, oral compositions areadministered with little or no regard to the timing of food intake.

Compositions for oral administration can be formulated as solid orliquid preparation. In some embodiments, a liquid formulation such assyrup, injection, eye drops or the like, is prepared with a pH adjustor(e.g., hydrochloric acid), solubilizer, isotonizing agent or the like,as well as a solubilizing aid, stabilizer, buffering agent, suspendingagent, antioxidant, etc., if necessary. In some embodiments, a liquidformulation is lyophilized, and an injection is administeredintravenously, subcutaneously or intramuscularly. Suspending agents thatcan be used include, but not limited to, methyl cellulose, polysorbate80, hydroxyethyl cellulose, gum arabic, tragacanth powder, sodiumcarboxymethylcellulose, polyoxyethylene sorbitan monolaurate and thelike. Solubilizing aids that can be used include, but not limited to,polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide,polyoxyethylene sorbitan monolaurate and the like. Stabilizing agentsthat can be used include, but not limited to, t sodium sulfite, sodiummetasulfite, ether and the like. Preservatives that can be used include,but not limited to, methyl paraoxybenzoate, ethyl paraoxybenzoate,sorbic acid, phenol, cresol, chlorocresol, and the like.

In some embodiments, provided compositions may be formulated for rectaladministration, e.g., as suppositories. Such rectally-appropriate formscan be prepared, for example, by mixing the agent with a suitablenon-irritating excipient that is solid at room temperature but liquid atrectal temperature and therefore will melt in the rectum to release thedrug. Such materials include cocoa butter, beeswax and/or polyethyleneglycols.

In some embodiments, provided compositions are formulated for topicaladministration, for example, the treatment site includes areas or organsreadily accessible by topical application, for example, the eye, theskin, or the lower intestinal tract.

Topical application to the lower intestinal tract can often be effectedwith a rectal suppository formulation (see above) or in a suitable enemaformulation. In some embodiments, topical or transdermal patches may beused.

In some embodiments, topical formulations are prepared in a suitableointment containing an active component suspended or dissolved in one ormore carriers. Carriers for topical administration typically include,but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Topical compositions can beformulated in a suitable lotion or cream, for example, containing one ormore active components suspended or dissolved in one or morepharmaceutically acceptable carriers. Suitable carriers may include, butare not limited to, mineral oil, sorbitan monostearate, polysorbate 60,cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol andwater, and combinations thereof.

Formulations for ophthalmic delivery are often prepared as solutions orsuspensions (e.g., isotonic, pH adjusted sterile saline). In someembodiments, one or more preservatives (e.g., benzylalkonium chloride)is/are also included. Ophthalmic compositions may be formulated in anointment such as petrolatum.

Compositions for nasal delivery are commonly formulated as aerosols.Such aerosol formulations may, for example, be or include solutions orsuspensions (e.g., in saline), optionally containing one or morepreservatives (e.g., benzyl alcohol), absorption promoters (e.g., toenhance bioavailability), and/or solubilizing or dispersing agents(e.g., fluorocarbons).

In some embodiments, compositions (e.g., pharmaceutical compositions) asdescribed herein may include one or more processing agents and/orcrystallization inhibitors, or combinations thereof.

In some embodiments, provided compositions contain one or moreprocessing agents. In some embodiments, the processing agent is water.In some embodiments, the processing agent is tert-butyl alcohol. In someembodiments, the processing agent is talc. In some embodiments, theprocessing agent is lactose. In some embodiments, the processing agentis precipitated calcium carbonate. In some embodiments, the processingagent is titanium dioxide. In some embodiments, the processing agent issilica. In some embodiments, the processing agent is microcrystallinecellulose.

In some embodiments, provided compositions comprise one or morecrystallization inhibitors. In some embodiments, the crystallizationinhibitor is water soluble. In certain embodiments, the crystallizationinhibitor is water insoluble.

Exemplary crystallization inhibitors include, but are not limited to,polyvinylpyrrolidone (PVP or povidone), including homo- and copolymersof polyvinylpyrrolidone and homopolymers or copolymers ofN-vinylpyrrolidone; crospovidone; gums; cellulose derivatives (e.g.,HPMC polymers, hydroxypropyl cellulose, ethyl cellulose,hydroxyethylcellulose, sodium carboxymethyl cellulose, calciumcarboxymethyl cellulose, sodium carboxymethyl cellulose); dextran;acacia; homo- and copolymers of vinyllactam, and mixtures thereof;cyclodextrins; gelatins; hypromellose phthalate; sugars; sugar alcoholsincluding mannitol; polyhydric alcohols; polyethylene glycol (PEG);polyethylene oxides; polyoxyethylene derivatives; polyvinyl alcohol;propylene glycol derivatives and the like, SLS, Tweens, Eudragits(methacrylic acid copolymers); and combinations thereof; amino acidssuch as prolin.

In some embodiments, the Compound I in the composition is amorphous. Insome embodiments, the crystallization inhibitor is polyvinylpyrrolidone(PVP or povidone). In some embodiments, the crystallization inhibitor ispovidone USP/NF, Ph. Eur, or JPE. In some embodiments, the amount ofCompound I and the amount of povidone is present in a composition in aratio of about 1:2 (by weight). In some embodiments, the amount ofCompound I and the amount of povidone is present in a composition in aratio of about 1:1 (by weight). In some embodiments, the amount ofCompound I and the amount of povidone is present in a composition in aratio of about 2:1 (by weight). In some embodiments, the amount ofCompound I and the amount of povidone is present in a composition in aratio of about 3:1 (by weight). In some embodiments, the amount ofCompound I and the amount of povidone is present in a composition in aratio of about 4:1 (by weight)). In some embodiments, the amount ofCompound I and the amount of povidone is present in a composition in aratio of about 5:1 (by weight).

In certain embodiments, a crystallization inhibitor employed by thepresent disclosure is a PVP polymer.

In certain embodiments, PVP polymers employed in the present disclosurehave a molecular weight of about 2,000 to about 50,000 Daltons, about2,000 to about 30,000 Daltons, about 2,000 to about 20,000 Daltons,about 2,500 to about 15,000 Daltons, about 2,500 to about 10,000Daltons, or about 3,000 to about 10,000 Daltons.

In certain embodiments, PVP polymers employed in the present disclosurehave a dynamic viscosity (10% in water at 20° C.) of about 1.3 to about700, about 1.5 to about 500, about 1.8 to about 300, about 2.0 to about200, about 2.2 to about 150, about 2.5 to about 100, about 2.8 to about70, about 3.0 to about 40, about 3.2 to about 25, or about 3.5 to about8.5 mPas.

Any type of povidone can be used in the compositions provides herein. Insome embodiments, povidone is selected from Plasdone® PVP polymers,which are synthetic, water-soluble homopolymers ofN-vinyl-2-pyrrolidone. Plasdone polymers useful in the compositionsprovided herein include, but are not limited to, Plasdone C-12 andPlasdone C-17.

In some embodiments, povidone possesses K values between 12 and 17. Insome embodiments, povidone possesses K values between 12 and 15.

In certain embodiments, PVP polymers employed in the present disclosureare selected from Kollidon® PVP polymers (e.g., Kollidon® 12PF,Kollidon® 17PF).

In certain embodiments, a crystallization inhibitor employed by thepresent disclosure is a PEG polymer.

In certain embodiments, PEG polymers employed in the present disclosurehave has an average molecular about 5,000-20,000 Dalton, about5,000-15,000 Dalton, or about 5,000-10,000 Dalton.

In certain embodiments, a crystallization inhibitor employed by thepresent disclosure is a surfactant. In certain embodiments, thecrystallization inhibitor is a Tween®surfactant. Exemplary Tweens®include Tween®20, Tween®40, Tween®60, Tween®65 and Tween®80.

In certain embodiments, a crystallization inhibitor employed by thepresent disclosure is an HPMC (hydroxypropylmethyl cellulose) polymer.

HPMC polymers vary in the chain length of their cellulosic backbone andconsequently in their viscosity as measured for example at a 2% (w/w) inwater. In certain embodiments, the HPMC polymer has a viscosity in water(at a concentration of 2% (w/w)), of about 100 to about 100,000 cP,about 1000 to about 15,000 cP, for example about 4000 cP. In certainembodiments, the molecular weight of the HPMC polymer has greater thanabout 10,000, but not greater than about 1,500,000, not greater thanabout 1,000,000, not greater than about 500,000, or not greater thanabout 150,000.

HPMC polymers also vary in the relative degree of substitution ofavailable hydroxyl groups on the cellulosic backbone by methoxy andhydroxypropoxy groups. With increasing hydroxypropoxy substitution, theresulting HPMC polymer becomes more hydrophilic in nature. In certainembodiments, the HPMC polymer has about 15% to about 35%, about 19% toabout 32%, or about 22% to about 30%, methoxy substitution, and havingabout 3% to about 15%, about 4% to about 12%, or about 7% to about 12%,hydroxypropoxy substitution.

Exemplary HPMC polymers include, but are not limited to,hydroxypropylmethylcellulose (HPMC), hydroxypropylmethylcelluloseacetate phthalate (HPMC-AP), hydroxypropylmethylcellulose acetatesuccinate (HPMC-AS), hydroxypropylmethylcellulose acetate trimellitate(HPMC-AT) and hydroxypropylmethylcellulose phthalate (HPMC-P).

Grades of hydroxypropylmethylcellulose (HPMC) include, but are notlimited to, 3FG, 4FG, 5FG, 6FG, 15FG, 50FG and K100M. Grades ofhydroxypropylmethylcellulose acetate succinate (HPMC-AS) includeHPMC-AS-LF, HPMC-AS-MF, HPMC-AS-HF, HPMC-AS-LG, HPMC-AS-MG andHPMC-AS-HG. Grades of hydroxypropyl-methylcellulose phthalate (HPMC-P)include 50, 55, 55S.

Other exemplary HPMC polymers are available under the brand namesMethocel™ of Dow Chemical Co. and Metolose™ of Shin-Etsu Chemical Co.Examples of suitable HPMC polymers having medium viscosity includeMethocel™ E4M, and Methocel™ K4M, both of which have a viscosity ofabout 4000 cP at 2% (w/w) water. Examples of HPMC polymers having higherviscosity include Methocel™ E10M, Methocel™ K15M, and Methocel™ K100M,which have viscosities of about 10,000 cP, 15,000 cP, and 100,000 cPrespectively viscosities at 2% (w/w) in water.

In some embodiments, provided formulation may include one or morecrystallization inhibitors. In certain embodiments, the secondcrystallization inhibitor is a PVP polymer. In certain embodiments, thesecond crystallization inhibitor is a PEG polymer. In certainembodiments, the second crystallization inhibitor is a Tween®surfactant. In certain embodiments, the formulation or compositioncomprises an amount of one or more crystallization inhibitors of atleast about 1%, 5%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% (w/w), based onthe total weight of the formulation or composition.

In some embodiments, the composition is prepared by lyophilization froma solution. In particular embodiments, the composition is prepared bylyophilization from a solution of (60:40) (v/v) t-butanol/water. In someembodiments, the solvent is tert-butanol. In some embodiments, thesolvent is a mixture of tert-butanol and water. In some embodiments, thepH adjustor is hydrochloric acid.

ISTODAX® Label

ISTODAX® is supplied as a kit which includes a sterile, lyophilizedpowder in a single-use vial containing 10 mg of Compound I and 20 mg ofthe bulking agent, povidone, USP. Additionally, each kit includes 1sterile vial containing 2 mL of the Diluent composed of 80% propyleneglycol, USP, and 20% dehydrated alcohol, USP.

The K value of Povidone USP is 17. The molecular weight of povidone USPis about 10.000 Dalton.

ISTODAX® is administered at a dose of 14 mg/m² intravenously over a4-hour period on days 1, 8 and 15 of a 28-day cycle. Cycles are repeatedevery 28 days.

Uses

Conditions to be Treated

Provided are methods and compositions relating to treatment of cellproliferative disorders, diseases or conditions. Cell proliferativedisorders, diseases or conditions include a variety of conditionscharacterized by aberrant cell growth, preferably abnormally increasedcellular proliferation. Cell proliferative disorders, diseases, orconditions that can be treated using the provided compositions andmethods include, but are not limited to, cancer, immune-mediatedresponses and diseases (e.g., transplant rejection, graft vs. hostdisease, immune reaction to gene therapy, autoimmune diseases,pathogen-induced immune dysregulation, etc.), certain circulatorydiseases, and certain neurodegenerative diseases.

In certain embodiments, provided are methods of treating cancer. Canceris a group of diseases which are characterized by uncontrolled growthand spread of abnormal cells. Cancers include, but are not limited to,carcinomas, sarcomas, leukemias, lymphomas and the like. In certainembodiments, cancer is a hematological malignancy. In certainembodiments, cancer is a solid tumor.

In certain embodiments the present disclosure relates to treatment ofhematological malignancies. Manifestations of hematological malignanciesinclude circulating malignant cells and malignant masses. Hematologicalmalignancies are types of cancers that affect the blood, bone marrow,and/or lymph nodes. Hematological malignancies that may be treated usingromidepsin include, but are not limited to: acute lymphoblastic leukemia(ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia(CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma(CTCL), peripheral T-cell lymphoma (PTCL), multiple myeloma, andmyelodysplastic syndromes. In certain embodiments, romidepsin is used totreat multiple myeloma. In certain particular embodiments, the cancer isrelapsed and/or refractory multiple myeloma. In other embodiments,romidepsin is used to treat chromic lymphocytic leukemia (CLL). Incertain particular embodiments, the cancer is relapsed and/or refractoryCLL. In other embodiments, romidepsin is used to treat chromicmyelogenous leukemia (CML). In certain embodiments, romidepsin is usedto treat acute lymphoblastic leukemia (ALL). In certain embodiments,romidepsin is used to treat acute myelogenous leukemia (AML). In certainembodiments, the cancer is cutaneous T-cell lymphoma (CTCL). In otherembodiments, the cancer is peripheral T-cell lymphoma (PTCL). In certainembodiments, the cancer is a myelodysplastic syndrome.

In some embodiments of the present disclosure, cancers treated include,but are not limited to, leukemias and lymphomas such as cutaneous T-celllymphoma (CTCL), peripheral T-cell lymphoma, lymphomas associated withhuman T-cell lymphotropic virus (HTLV) such as adult T-cellleukemia/lymphoma (ATLL), B-cell lymphomas, acute lymphocytic leukemia,acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronicmyelogenous leukemia, acute myelogenous leukemia, Hodgkin's disease,non-Hodgkin's lymphomas, multiple myeloma, myelodysplastic syndromes.

In some such embodiments the disclosure relates to treatment of solidtumors such as lung, breast, colon, liver, pancreas, renal, prostate,ovarian, and/or brain. In some embodiments, the disclosure relates totreatment of pancreatic cancer. In some embodiments, the disclosurerelates to treatment of renal cancer. In some embodiments, thedisclosure relates to treatment of prostate cancer. In some embodiments,the disclosure relates to treatment of sarcomas. In some embodiments,the disclosure relates to treatment of soft tissue sarcomas.

In some embodiments, cancers that can be treated are solid cancers thatinclude, but are not limited to, mesothelioma, common solid tumors ofadults such as head and neck cancers (e.g., oral, laryngeal andesophageal), genitourinary cancers (e.g., prostate, bladder, renal,uterine, ovarian, testicular, rectal and colon), melanoma and other skincancers, stomach cancer, brain tumors, liver cancer and thyroid cancer,and/or childhood solid tumors such as brain tumors, neuroblastoma,retinoblastoma, Wilms' tumor, bone tumors, and soft-tissue sarcomas. Insome embodiments, the disclosure relates to treatment of solid tumors.

Cancers that may be treated using the methods provided herein, includingcombination therapy, include but not limited to, colon cancer, lungcancer, bone cancer, pancreatic cancer, stomach cancer, esophagealcancer, skin cancer, brain cancer, liver cancer, ovarian cancer,cervical cancer, uterine cancer, testicular cancer, prostate cancer,bladder cancer, kidney cancer, and neuroendocrine cancer.

In certain embodiments, cancer is pancreatic cancer. In certainembodiments, cancer is prostate cancer. In certain specific embodiments,the prostate cancer is hormone refractory prostate cancer.

In some particular embodiments, provided are methods to treat leukemias.In some embodiments, leukemia is chronic lymphocytic leukemia, chronicmyelogenous leukemia, acute lymphocytic leukemia, acute myelogenousleukemia, or adult T cell leukemia/lymphoma.

In some embodiments, provided are methods of treating lymphomas. In someembodiments, lymphoma is Hodgkin's or non-Hodgkin's (e.g., T-celllymphomas such as peripheral T-cell lymphoma, cutaneous T-cell lymphoma,etc.) lymphoma.

In some embodiments, the disclosure relates to the treatment of multiplemyeloma and/or myelodysplastic syndromes.

In some embodiments, provided are methods of treating one or moreimmune-mediated responses and diseases including, but not being limitedto, rejection following transplantation of synthetic or organic graftingmaterials, cells, organs, or tissue to replace all or part of thefunction of tissues, such as heart, kidney, liver, bone marrow, skin,cornea, vessels, lung, pancreas, intestine, limb, muscle, nerve tissue,duodenum, small-bowel, pancreatic-islet-cell, includingxeno-transplants, etc.; graft vs host disease; autoimmune diseases, suchas rheumatoid arthritis, systemic lupus erythematosus, thyroiditis,Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type Idiabetes, juvenile-onset or recent-onset diabetes mellitus, uveitis,Graves' disease, psoriasis, atopic dermatitis, Crohn's disease,ulcerative colitis, vasculitis, auto-antibody mediated diseases,aplastic anemia, Evan's syndrome, autoimmune hemolytic anemia, and thelike.

In some embodiments, provided are methods of treating of one or moreinfectious diseases causing aberrant immune response and/or activation,such as traumatic or pathogen induced immune dysregulation, includingfor example, that which are caused by hepatitis B and C infections, HIV,Staphylococcus aureus infection, viral encephalitis, sepsis, parasiticdiseases wherein damage is induced by an inflammatory response (e.g.,leprosy).

In some embodiments, provided are methods of treatment of graft vs hostdisease, rheumatoid arthritis, systemic lupus erythematosus, psoriasis,atopic dermatitis, Crohn's disease, ulcerative colitis, or multiplesclerosis.

In some embodiments, provided are methods of treatment of an immuneresponse associated with a gene therapy treatment, such as theintroduction of foreign genes into autologous cells and expression ofthe encoded product. In some embodiments, provided are methods oftreating of circulatory diseases, such as arteriosclerosis,atherosclerosis, vasculitis, polyarteritis nodosa or myocarditis.

In some embodiments, provided are methods of treatment of any of avariety of neurodegenerative diseases, a non-exhaustive list of whichincludes:

I. Disorders characterized by progressive dementia in the absence ofother prominent neurologic signs, such as Alzheimer's disease; Seniledementia of the Alzheimer type; and Pick's disease (lobar atrophy);

II. Syndromes combining progressive dementia with other prominentneurologic abnormalities such as: A) syndromes appearing mainly inadults (e.g., Huntington's disease, Multiple system atrophy combiningdementia with ataxia and/or manifestations of Parkinson's disease,Progressive supranuclear palsy (Steel-Richardson-Olszewski), diffuseLewy body disease, and corticodentatonigral degeneration); and B)syndromes appearing mainly in children or young adults (e.g.,Hallervorden-Spatz disease and progressive familial myoclonic epilepsy);

III. Syndromes of gradually developing abnormalities of posture andmovement such as paralysis agitans (Parkinson's disease), striatonigraldegeneration, progressive supranuclear palsy, torsion dystonia (torsionspasm; dystonia musculorum deformans), spasmodic torticollis and otherdyskinesis, familial tremor, and Gilles de la Tourette syndrome;

IV. Syndromes of progressive ataxia such as cerebellar degenerations(e.g., cerebellar cortical degeneration and olivopontocerebellar atrophy(OPCA)); and spinocerebellar degeneration (Friedreich's ataxia andrelated disorders);

V. Syndromes of central autonomic nervous system failure (Shy-Dragersyndrome);

VI. Syndromes of muscular weakness and wasting without sensory changes(motomeuron disease such as amyotrophic lateral sclerosis, spinalmuscular atrophy (e.g., infantile spinal muscular atrophy(Werdnig-Hoffman), juvenile spinal muscular atrophy(Wohlfart-Kugelberg-Welander) and other forms of familial spinalmuscular atrophy), primary lateral sclerosis, and hereditary spasticparaplegia;

VII. Syndromes combining muscular weakness and wasting with sensorychanges (progressive neural muscular atrophy; chronic familialpolyneuropathies) such as peroneal muscular atrophy(Charcot-Marie-Tooth), hypertrophic interstitial polyneuropathy(Dejerine-Sottas), and miscellaneous forms of chronic progressiveneuropathy;

VIII. Syndromes of progressive visual loss such as pigmentarydegeneration of the retina (retinitis pigmentosa), and hereditary opticatrophy (Leber's disease).

In some embodiments, the neurodegenerative disease is Alzheimer'sdisease, Parkinson's disease, and/or Huntington's disease.

In some embodiments, the diseases or conditions are associated withchromatin remodeling.

Dosing

In some embodiments, Compound I and/or compositions containing CompoundI is/are administered according to a standard dosing regimen. In someembodiments, Compound I and/or compositions containing Compound I is/areadministered according to an accelerated dosing regimen.

Standard Dosing for Compound I

In some embodiments, unit doses of Compound I are within the range ofabout 0.5 mg/m² to about 28 mg/m² body surface area. In someembodiments, the range of about 6 to about 18 mg/m² is used. In someembodiments, the range is about 10 mg/m² to about 17 mg/m². In someembodiments, particular unit doses are 10 mg/m², 12 mg/m², 13 mg/m², 14mg/m², and 15 mg/m².

In some embodiments, Compound I is administered intravenously. In someembodiments, intravenous dosing regimens include daily dosing for 2weeks, twice weekly dosing for 4 weeks, thrice weekly dosing for 4weeks, and various other intermittent schedules (e.g., on days 1, 3, and5; on days 4 and 10; on days 1, 8 and 15; on days 1 and 15; on days 5and 12; or on days 5, 12, and 19 of 21 or 28 day cycles).

In some embodiments, Compound I is administered in individual unit dosesover 4 hours on days 1, 8, and 15, with courses repeating every 28 days.Often, several courses (e.g., at least 4, at least 6, or more) areadministered. Indeed, instances have been reported of as many as 72courses being administered. In some embodiments, individual unit dosesare administered by 4 hour infusion.

Accelerated Dosing for Compound I

Accelerated dosing regimens for Compound I may be utilized, in which oneor more individual unit doses is administered intravenously over aperiod of time that is less than or equal to about one hour. In someembodiments, one or more individual doses are administered intravenouslyover a period of time that is less than about 50 minutes, 40 minutes, 30minutes, 20 minutes, or less. Any regimen that includes at least oneunit dose administered over a period of time that is less than about onehour (60 minutes) may be considered an accelerated dosing regimen inaccordance with the present disclosure.

In some embodiments, all unit doses within a regimen are administeredintravenously over a time period that is less than or equal to about onehour. In some embodiments, only some of the unit doses within a regimenare administered over a time period that is less than or equal to aboutone hour. In some embodiments, one or more unit doses within a regimenare administered by a route other than intravenous administration (e.g.,oral, subcutaneous, nasal, topical, etc).

Accelerated dosing regimens of Compound I can be administered without asignificant increase in toxicity or adverse events, particularly inserious adverse events, as compared with a comparable regimen (e.g., anotherwise identical regimen) in which individual unit doses areadministered intravenously over a 4-hour period. Accelerated dosingregimens can be administered without a significant increase in toxicityor adverse events, particularly in serious adverse events, as comparedwith a standard regimen of Compound I administered by 4-hour intravenousinfusion of a dose of about 6-14 mg/m² on days 1, 8, and 15 of a 28 daycycle.

In some embodiments, Compound I is administered in an accelerated dosingregimen that is identical to a standard dosing regimen (see above)except that one or more unit doses is administered over a time periodthat is less than about 1 hour (e.g., rather than over a time period ofabout 4 hours).

In some embodiments, unit doses of Compound I are within the range ofabout 0.5 mg/m² to about 28 mg/m². In certain embodiments, unit dosesare in the range of about 1 mg/m² to about 25 mg/m². In certainembodiments, unit doses are in the range of about 0.5 mg/m² to about 15mg/m². In certain embodiments, unit doses are the range of about 1 mg/m²to about 15 mg/m². In certain embodiments, unit doses are in the rangeof about 1 mg/m² to about 8 mg/m². In certain embodiments, unit dosesare in the range of about 0.5 mg/m² to about 5 mg/m². In certainembodiments, the unit doses are in the range of about 2 mg/m² to about10 mg/m². In some embodiments, unit doses are in the range of about 10mg/m² to about 20 mg/m². In certain embodiments, unit doses are in therange of about 5 mg/m² to about 10 mg/m². In some embodiments, unitdoses are in the range of about 10 mg/m² to about 15 mg/m². In someembodiments, unit doses are in the range of about 6 to about 19 mg/m².In some embodiments, unit doses are approximately 8 mg/m². In stillother embodiments, the unit doses are approximately 9 mg/m². In stillother embodiments, unit doses are approximately 10 mg/m². In still otherembodiments, unit doses are approximately 11 mg/m². In still otherembodiments, unit doses are approximately 12 mg/m². In still otherembodiments, unit doses are approximately 13 mg/m². In still otherembodiments, unit doses are approximately 14 mg/m². In still otherembodiments, unit doses are approximately 15 mg/m². In still otherembodiments, unit doses are approximately 30 mg/m².

In certain embodiments, different individual unit doses within aCompound I therapy regimen are different. In some embodiments,increasing doses of Compound I are administered over the course of acycle. In certain embodiments, a dose of approximately 8 mg/m² isadministered, followed by a dose of approximately 10 mg/m², followed bya dose of approximately 12 mg/m² may be administered over a cycle.

An amount of Compound I administered in individual unit doses variesdepending on the form of Compound I being administered. The dosagesgiven herein are dose equivalents with respect to the active ingredient,Compound I.

In certain embodiments, individual unit doses of Compound I areadministered on one day followed by several days on which Compound I isnot administered. In certain embodiments, Compound I is administeredtwice a week. In certain embodiments, Compound I is administered once aweek. In other embodiments, Compound I is administered every other week.

In some embodiments, Compound I is administered daily (for example for 2weeks), twice weekly (for example for 4 weeks), thrice weekly (forexample for 4 weeks), or on any of a variety of other intermittentschedules (e.g., on days 1, 3, and 5; on days 4 and 10; on days 1 and15; on days 5 and 12; or on days 5, 12, and 19 of 21 or 28 day cycles).

In certain embodiments, Compound I is administered on days 1, 8, and 15of a 28 day cycle. In certain particular embodiments, an 8 mg/m² dose ofCompound I is administered on day 1, a 10 mg/m² dose of Compound I isadministered on day 8, and a 12 mg/m² dose of Compound I is administeredon day 15. In certain embodiments, Compound I is administered on days 1and 15 of a 28 day cycle with day 8 being skipped. A 28 day dosing cyclemay be repeated. In certain embodiments, a 28 day cycle is repeated2-10, 2-7, 2-5, or 3-10 times. In certain embodiments, the treatmentincludes 5 cycles. In certain embodiments, the treatment includes 6cycles. In certain embodiments, the treatment includes 7 cycles. Incertain embodiments, the treatment includes 8 cycles. In certainembodiments, 10 cycles are administered. In certain embodiments, greaterthan 10 cycles are administered.

In certain embodiments, one or more unit doses within a Compound Idosing regimen may be administered via a route other than intravenousadministration. In some embodiments, one or more doses may beadministered orally. In certain embodiments, Compound I is dosed orallyin the range of 10 mg/m² to 300 mg/m². In certain embodiments, CompoundI is dosed orally in the range of 25 mg/m² to 100 mg/m². In certainembodiments, Compound I is dosed orally in the range of 100 mg/m² to 200mg/m². In certain embodiments, Compound I is dosed orally in the rangeof 200 mg/m² to 300 mg/m². In certain embodiments, Compound I is dosedorally at greater than 300 mg/m². In certain embodiments, Compound I isdosed orally in the range of 50 m g/m² to 150 mg/m². In otherembodiments, the oral dosage ranges from 25 mg/m² to 75 mg/m².

In certain embodiments, Compound I is administered orally on a dailybasis. In some embodiments, Compound I is administered orally everyother day. In still other embodiments, Compound I is administered orallyevery third, fourth, fifth, or sixth day. In certain embodiments,Compound I is administered orally every week. In certain embodiments,Compound I is administered orally every other week.

In some embodiments, one or more unit doses of Compound I isadministered topically.

As will be appreciated by one of skill in the art, the dosage, timingand/or routes of administration of particular unit doses of Compound Imay vary depending on the patient and condition being treated. Incertain embodiments, the cycles are continued as long as the patient isresponding. Therapy may be terminated once there is disease progression,a cure or remission is achieved, or side effects become intolerable.Adverse side effects may also call for lowering the dosage of Compound Iadministered, or for adjusting the schedule by which doses areadministered.

Toxicity and Adverse Events with Compound I

Compound I has been administered to patients in a variety of differentclinical contexts and studies. Observed toxicities include fatigue,nausea, vomiting, and myelosuppression (thrombocytopenia and/orneutropenia, e.g., Grade 3). Non-specific S-T segment changes on ECG andprolongation of QTc intervals occur in many patients. Observedtoxicities were mild to moderate. Observed changes in ECGs did notcorrelate with elevated serial serum troponin levels and multiple gatedacquisition (MUGA) scans, both of which were consistently normal.

In early development, 6 deaths occurred (out of more than 450 patients)during clinical investigations of Compound I. In all but one of thedeaths, significant cardiovascular risk factors were either present atthe time of entry into the Compound I study or appeared during thecourse of the study. The sixth patient had a history of sarcoidosis andwas simultaneously administered another drug that also is known to causeQTc prolongation.

Hematologic Events

Administration of Compound I may cause neutropenia and/orthrombocytopenia It is generally recommended that further treatment bewithheld from patients with Grade 3 or Grade 4 neutropenia orthrombocytopenia, until their specific cytopenia returns to Grade 1(i.e., ANC recovered to >1.9×10⁹/L and platelet count recovered to≧75×10⁹/L) or below, at which point therapy can be continued at fulldose. If Grade 4 neutropenia or thrombocytopenia lasting more than 5days or associated with bleeding, then it is generally recommended thattreatment be withheld until specific cytopenia returns to Grade 1 orbelow, at which point therapy can continue, preferably at a reduced dose(e.g., 10 mg/m²). If Grade 4 febrile (≧38.5° C.) neutropenia orthrombocytopenia that requires platelet transfusion is observed, it isgenerally recommended that treatment be withheld until the specificcytopenia returns to Grade 1 or below, at which point therapy cancontinue, preferably at a reduced dose (e.g., 10 mg/m²).

Hematologic events are typically observed at a rate of about 21-52% withstandard Compound I dosing regimens (National Cancer Institute IND57,810 Annual Report, 2007). For example, the NCI-2007 Annual Reportprovides the following rates for the following blood and bone marrowabnormalities: platelets (52%), hemoglobin/anemia (41%), abnormal whiteblood cell count (39%), abnormal ANC/AGC (37%), and lymphopenia (21%)(National Cancer Institute IND 57,810 Annual Report, 2007).

Cardiac Events

Cardiac events observed with Compound I administration can include anyor all of the following:

Prolongation of QTc to ≧500 msec or an increase of ≧60 msec frompretreatment baseline for the current treatment cycle;

Ventricular arrhythmia (i.e., ventricular tachycardia or ventricularfibrillation [≧3 beats in a row)′

Sinus tachycardia (pulse >140/min after recumbency);

New occurrence of atrial dysrhythmias (SVT, atrial fibrillation, oratrial flutter), ST and T-wave changes indicative of repolarizationabnormalities or ischemia (e.g., ST depression of ≧2 mm [measured fromisoelectric line to ST segment] and/or T-wave inversion of ≧4 mm[measured from isoelectric line to peak of T-wave] as long as the mainQRS vector is positive).

The literature reports that the median change in QTc from baseline is16.5 milliseconds (see, Piekarz et al., Clin Cancer Res 12:3762, 2006).Table 2 presents common recommendations for dose modification whencardiac events are observed.

TABLE 2 Recommendation for dose modification during cardiac eventsParameters/Symptoms Change Action Dosing/Continuation Sinus tachycardiaPulse > 140/min after Hold further dosing, If resolved, restartrecumbancy consult local treatment, preferably at a Atrial dysrhythmia(SVT, New occurrence cardiologist, and treat reduced dose (e.g., 10atrial fibrillation, or atrial appropriately mg/m²) flutter) If notresolved, Prolongation of QTcf To ≧500 msec discontinue therapy comparedto pre-treatment OR baseline in a treatment Increase by ≧60 msec cycleVentricular tachycardia ≧3 beats in a row Ventricular fibrillation Newoccurrence Hold further dosing and Hold further dosing until treatappropriately. The medical monitor and medical monitor shouldcardiologist evaluation is be notified and local complete cardiologistshould be consulted A subsequent episode of any of the above, despitedose reduction Discontinue Compound I administration T-wave morphologyInversion of ≧4 mm^(a) Hold further dosing, If resolved, restartST-segment Depression of ≧2 mm^(b) consult local treatment, preferablyat a cardiologist, and treat reduced dose (e.g., 10 appropriately mg/m²)In some patients, ST segment and T-wave morphology changes may recurdespite a dose reduction. In such cases, further treatment should bewithheld until the ECG changes resolve. If the patient experiences noconcomitant clinical events, treatment may be resumed, preferably at thereduced dose level. If not resolved, discontinue therapy. ^(a)Measuredfrom isoelectric line to peak of T-wave ^(b)Measured from isoelectricline to ST segment

Cardiac events are typically observed at a rate of about 24% withstandard Compound I dosing regimens (National Cancer Institute IND57,810 Annual Report, 2007).

Gastrointestinal Events

Gastrointestinal events are typically observed at a rate of about 15-64%with standard Compound I dosing regimens (National Cancer Institute IND57,810 Annual Report, 2007). For example, the NCI-2007 Annual Reportprovides the following rates for the following gastrointestinal events:nausea (64%), anorexia (39%), vomiting (39%), constipation (19%),dysguesia (18%), and diarrhea (15%) (National Cancer Institute IND57,810 Annual Report, 2007).

Compound I can be administered via accelerated dosing regimens without aclinically significant increase in relevant toxicities (e.g., in therate and/or severity of one or more of dose limiting toxicities, seriousadverse events, and/or adverse events). In some embodiments, providedare accelerated dosing regimens for Compound I in which the rate ofobserved toxicities (e.g., fatigue, hematological toxicities, cardiactoxicities, gastrointestinal toxicities, constitutional toxicities, or acombination thereof) is not materially worse than that observed foradministration of a comparable dosing regimen that differs only in thatunit doses of Compound I are administered intravenously over a timeperiod of about 4 hours. In some embodiments, provided are accelerateddosing regimens for Compound I in which the rate of observed toxicitiesis not materially worse than that observed for administration of astandard Compound I therapy regimen.

In some embodiments, provided are accelerated dosing regimens forCompound I in which the subject receiving Compound I does not suffer oneor more particular adverse events, or serious adverse events, within adesignated time period. In some embodiments, the designated time periodis during administration of the accelerated dose. In some embodiments,the designated time period is within about 2 to about 6 hours after theend of infusion of the accelerated dose. In some embodiments, thedesignated time period is within about 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 42, 44, 46, 48 or more hoursafter the end of infusion of the accelerated dose.

Any side effect, toxicity, or adverse event may be absent from thedesignated time period. In some embodiments, the subject's QTc remainsbelow about 500 msec during the designated time period; in someembodiments, the subject does not suffer a ventricular arrhythmia duringthe designated time period; in some embodiments, the subject does notsuffer sinus tachycardia during the designated time period; in someembodiments, the subject does not suffer an atrial dysrhythmia duringthe designated time period; in some embodiments the subject does notsuffer ST or T-wave changes indicative of repolarization during thedesignated time period.

Combination Therapy

In some embodiments, Compound I is administered in combination with oneor more other pharmaceutical agents. In some embodiments, Compound I isadministered in combination with one or more other chemotherapeuticagents and/or in combination with one or more other pharmaceuticalagents (e.g., pain relievers, anti-inflammatories, antibiotics,steroidal agents, anti-folates, kinase inhibitors, methyl transferaseinhibitors, antibodies, etc.).

In certain embodiments, Compound I is administered in combination withone or more cytotoxic agents. Exemplary cytotoxic agents include, butare not limited to, gemcitabine, decitabine, and flavopiridol. Incertain embodiments, Compound I is administered in combination with oneor more taxanes and/or one or more proteasome inhibitors. Exemplaryproteasome inhibitors include, but are not limited to, bortezomib(VELCADE®), peptide boronates, salinosporamide A (NPI-0052),lactacystin, epoxomicin (Ac(Me)-Ile-Ile-Thr-Leu-EX), MG-132(Z-Leu-Leu-Leu-al), PR-171, PS-519, eponemycin, aclacinomycin A,CEP-1612, CVT-63417, PS-341 (pyrazylcarbonyl-Phe-Leu-boronate), PSI(Z-Ile-Glu (OtBu)-Ala-Leu-al), MG-262 (Z-Leu-Leu-Leu-bor), PS-273(MNLB), omuralide (clasto-lactacystin-β-lactone), NLVS(Nip-Leu-Leu-Leu-vinyl sulfone), YLVS (Tyr-Leu-Leu-Leu-vs),dihydroeponemycin, DFLB (dansyl-Phe-Leu-boronate), ALLN(Ac-Leu-Leu-Nle-al), 3,4-dichloroisocoumarin,4-(2-aminoethyl)-benzenesulfonyl fluoride, TMC-95A, gliotoxin, EGCG((−)-epigallocatechin-3-gallate), YU101 (Ac-hFLFL-ex), and combinationsthereof.

In certain embodiments, Compound I is administered in combination withone or more anti-folates. In some such embodiments, Compound I isadministered in combination with one or more of: folinic acid(leucovorin), methotrexate, pralatrexate, premextred, triazinate, orcombinations thereof.

In certain embodiments, Compound I is administered in combination withone or more kinase inhibitors (e.g., tyrosine kinase inhibitors). Insome embodiments, Compound I is administered in combination with one ormore antibodies that act as a kinase inhibitor. In some embodiments,Compound I is administered in combination with one or more of ABT-869,AC220, AZD7762, BIBW 2992, BMS-690154, CDKIAT7519, CYC116, ISIS3521,GSK690693, GSK-461364, MK-0457, MLN8054, MLN8237, MP470, ON 01910.Na,OSI-930, PHA-739358, R935788, SNS-314, TLN-232, XL147, XL228, XL281,XL418, or XL765.

In certain embodiments, Compound I is administered in combination withone or more methyl transferase inhibitors.

In certain embodiments, Compound I is administered in combination withone or more therapeutic antibodies. In some embodiments, Compound I isadministered in combination with one or more of: bevacizumab, cetuximab,dasatinib, erlotinib, geftinib, imatinib, lapatinib, nilotinib,panitumumab, pegaptanib, ranibizumab, sorafenib, sunitinib, trastuzumab,or any antibody that binds to an antigen bound by one of these moieties.

In some embodiments, Compound I is administered in combination with ananti-inflammatory agent, pain reliever, anti-nausea medication, oranti-pyretic. Anti-inflammatory agents useful in the methods providedherein include, but are not limited to, aspirin, ibuprofen, andacetaminophen, etc.

In certain embodiments, Compound I is administered in combination with asteroidal agent. In certain embodiments, Compound I is administered incombination with a steroidal agent selected from the group consisting ofalclometasone diproprionate, amcinonide, beclomethasone diproprionate,betamethasone, betamethasone benzoate, betamethasone diproprionate,betamethasone sodium phosphate, betamethasone sodium phosphate andacetate, betamethasone valerate, clobetasol proprionate, clocortolonepivalate, cortisol (hydrocortisone), cortisol (hydrocortisone) acetate,cortisol (hydrocortisone) butyrate, cortisol (hydrocortisone) cypionate,cortisol (hydrocortisone) sodium phosphate, cortisol (hydrocortisone)sodium succinate, cortisol (hydrocortisone) valerate, cortisone acetate,desonide, desoximetasone, dexamethasone, dexamethasone acetate,dexamethasone sodium phosphate, diflorasone diacetate, fludrocortisoneacetate, flunisolide, fluocinolone acetonide, fluocinonide,fluorometholone, flurandrenolide, halcinonide, medrysone,methylprednisolone, methylprednisolone acetate, methylprednisolonesodium succinate, mometasone furoate, paramethasone acetate,prednisolone, prednisolone acetate, prednisolone sodium phosphate,prednisolone tebutate, prednisone, triamcinolone, triamcinoloneacetonide, triamcinolone diacetate, triamcinolone hexacetonide, orcombinations thereof. In some embodiments, Compound I is administered incombination with dexamethasone.

In certain embodiments, Compound I is administered in combination withan agent to treat gastrointestinal disturbances such as nausea,vomiting, and diarrhea. Such agents may include anti-emetics,anti-diarrheals, fluid replacement, electrolyte replacement, etc.

In certain embodiments, Compound I is administered in combination withelectrolyte replacement or supplementation such as potassium, magnesium,and calcium. In certain embodiments, Compound I is administered incombination with electrolyte replacement or supplementation such aspotassium, magnesium.

In certain embodiments, Compound I is administered in combination withan anti-arrhythmic agent.

In certain embodiments, Compound I is administered in combination withan agent that increases the production of platelets.

In certain embodiments, Compound I is administered in combination withan agent to boost the production of blood cells. In certain embodiments,the agent is erythropoietin.

In some embodiments, Compound I is administered in combination with anagent to prevent hyperglycemia.

In certain embodiments, Compound I is administered with another HDAC orDAC inhibitor.

Electrolyte Supplementation

In some embodiments, electrolyte supplementation is administered tosubjects receiving Compound I therapy. Individuals with low electrolytelevels (e.g., low potassium and/or magnesium levels) are susceptible todevelopment of unwanted side effects if administered Compound I therapy(see, for example, published application No. US 2008/0124403, which isincorporated herein by reference).

Such patients may be particularly susceptible to development of cardiacrepolarization effects, including QTc prolongation (though potentiallywith no significant cardiac function changes), and/or cardiacdysrhythmias. Particular abnormalities that may be observed include anincrease in QTc interval and/or abnormalities of the ST segment (e.g.,ST segment depression) and/or the T-wave (e.g., T-wave flattening) onECG.

An individual with a potassium serum concentration below about 3.5mmol/L (3.5 mEq/L) and/or a serum magnesium concentration below about0.8 mml/L (1.95 mEq/L) suffers an increased risk of developing cardiacrepolarization effects and/or dysrhythmias.

Serum concentrations of potassium are generally considered to be“normal” when they are within the range of about 3.5-5.5 mEq/L or about3.5-5.0 mEq/L. It is often desirable to ensure that an individuals'serum potassium concentration is within these ranges prior to (and/orduring) administration of Compound I therapy.

Serum concentrations of magnesium are generally considered to be“normal” when they are within the range of about 1.5-2.5 mEq/L or about1.5-2.2 mEq/L or about 1.25-2.5 mEq/L or about 1.25-2.2 mEq/L. It isoften desirable to ensure that an individual's serum magnesiumconcentration is within these ranges prior to (and/or during)administration of Compound I therapy.

In some embodiments, an individual's serum potassium and/or magnesiumconcentration(s) is/are at the high end of the normal range prior to(and/or during) administration of Compound I therapy. In someembodiments, an individual's serum potassium concentration is at leastabout 3.8 mEq/L, 3.9 mEq/L, 4.0 mEq/L, or more prior to and/or duringadministration of Compound I therapy. In some embodiments, care is takennot to increase serum potassium concentration above about 5.0 mEq/L, 5.2mEq/L, or 5.5 mEq/L. In some embodiments, an individual's serummagnesium concentration is at least about 1.9 mEq/L or more prior toand/or during administration of Compound I therapy. In some embodiments,care is taken not to increase magnesium concentration above about 2.5mEq/L.

In some embodiments of the present disclosure, an individual's serumpotassium concentration is at least about 3.5 mEq/L (in some embodimentsat least about 3.8 mEq/L, 3.9 mEq/L, 4.0 mEq/L, or above) and theindividual's serum magnesium concentration is at least about 1.85 mEq/L(in some embodiments at least about 1.25 mEq/L, 1.35 mEq/L, 1.45 mEq/L,1.55 mEq/L, 1.65 mEq/L, 1.75 mEq/L, 1.85 mEq/L, 1.95 mEq/L, or above)prior to and/or during administration of Compound I therapy.

In some embodiments, electrolyte levels (e.g., potassium and/ormagnesium levels, optionally calcium levels) are assessed more than onceduring the course of Compound I therapy; in some embodiments, differentassessments are separated by a regular interval (e.g., 0.5 days or less,1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days,10 days, 11 days, 12 days, 13 days, 14 days, 1 month, 2 months, 3months, 4 months, 5 months, 6 months, etc.). In some embodiments,electrolyte levels are assessed prior to each administration of CompoundI.

An individual's serum potassium and/or magnesium and/or otherelectrolyte concentration(s) may be assessed by any available means. Forexample, samples may be collected from venous or arterial blood andprocessed for plasma or serum analysis. In some embodiments, venoussampling is utilized. Any available assay may be utilized forassessment. In some embodiments, potassium is measured by flamephotometry, direct potentiometry (see, for example, Koch et al., Clin.Chem. 29:1090, 1983), enzymatic methods (e.g., by using tryptophanase;see, for example, Kimura et al., Clin. Chem. 38:44, 1992), colorimetricmethods (e.g., using tetraphenyl borate), etc. In some embodiments,magnesium is measured by complexometric titration, flame emissionphotometry, atomic absorption spectophotometry, other spectrophotometrictechniques including enzymatic techniques and dye binding methods (e.g.,Magnon dye binding and bichromatic absorbance; see, for example, Barbouret al., Clin. Chem. 34:2103, 1988; elimination of interference bybilirubin; see, for example, Rehak et al., Clin. Chem 35:1031, 1989;etc.). In many embodiments, assays are performed in an automatedclinical chemistry analyzer (e.g., the Abbott ARCHITECT®, etc.).

Where both potassium and magnesium levels are assessed, they may beassessed separately or together. Assessment of potassium and/ormagnesium levels may be performed prior to, at the same time as, and/orafter initiation of Compound I therapy.

In some embodiments, if an individual is determined to have serumpotassium and/or magnesium concentration(s) that is/are below normal, orbelow the high end of normal as described herein, potassium and/ormagnesium supplementation is administered prior to, at the same time as,or after initiation of Compound I therapy. In some embodiments, CompoundI therapy is suspended or delayed until serum potassium and/or magnesiumlevels are increased. In some embodiments, Compound I therapy issuspended or delayed until serum potassium and/or magnesium levels areincreased to within the normal range, or to within the upper end of thenormal range. In some embodiments, Compound I therapy is suspended ordelayed until serum potassium concentration is above about 3.5 mEq/L; oris above about 3.8 mEq/L. In some embodiments, Compound I therapy issuspended or delayed until serum magnesium concentration is above about1.25 mEq/L; or is above about 1.8 mEq/L; or is above about 1.9 mEq/L. Insome embodiments, Compound I therapy is suspended or delayed until bothserum potassium and serum magnesium concentrations are increased asdescribed.

In some embodiments, electrolyte supplementation may be administeredprior to, concurrently with, and/or subsequent to initiation of CompoundI therapy, and may include potassium and/or magnesium supplementation.In some embodiments, electrolyte supplementation may includesupplementation of one or more electrolytes selected from the groupconsisting of sodium, potassium, chloride, calcium, magnesium,bicarbonate, phosphate, sulfate, and combinations thereof.

A variety of different potassium supplemental forms is available (see,for example, the web page at the following world-wide-web address:pdrhealth.com). For example, potassium supplements in the form ofpotassium chloride, potassium citrate, potassium gluconate, potassiumbicarbonate, potassium aspartate and/or potassium orotate can readily beobtained.

One of potassium supplemental forms is high-potassium (up to 800milligrams per serving), low-sodium vegetable juices. Some soft drinksare rich in potassium. Some soft drinks contain potassium gluconatewhich has a less bitter taste than some other potassium supplements.Salt substitutes are high in potassium.

Certain foods high in potassium such as raisins, figs, apricots,sardines, veal, bananas, avocado, and broccoli may be used as potassiumsupplements. Foods high in potassium may provide potassium that iseasily bioavailable and/or may reduce gastrointestinal side effectsassociated with the administration of potassium salts. The potassiumsupplement may also be provided as part of a multivitamin.

Potassium is typically supplemented orally or intravenously, thoughother modes of delivery are within the scope of the present disclosure.

Certain commercially available forms of potassium supplements include,for example, potassium acetate (e.g., 2 mEq/mL or 4 mEq/mL forinjection); potassium acetate (e.g., 75 mg, 95 mg, 99 mg, and 180 mgtablets and/or 2 mEq/mL, 10 mEq/50 mL, 20 mEq/50 mL, 10 mEq/100 mL, 20mEq/100 mL, 30 mEq/100 mL, 40 mEq/100 mL for injection and/or 20 mEq/15mL, 40 mEq/15 mL liquid and/or 20 mEq or 25 mEq powder forreconstitution, and/or 9 mEq, 10 mEq, or 20 mEq extended releasetablets), and potassium gluconate (e.g., 486 mg, 500 mg, 550 mg, 595 mg,610 mg, and 620 mg tablets).

A variety of different magnesium supplemental forms are also available.For example, supplements in the form of magnesium chloride, magnesiumgluconate, magnesium lactate, magnesium oxide and/or magnesium sulfatecan readily be obtained.

Certain foods high in magnesium such as artichoke, banana, figs,almonds, cashews, pine nuts, brazil nuts, beans, spinach, and tomatoesmay be used as magnesium supplements. The magnesium supplement may alsobe provided as part of a multivitamin.

Certain commercially available forms of magnesium supplements includemagnesium chloride (e.g., 200 mg/ml for injection, 535 mg extendedrelease tablets), magnesium gluconate (3.25 mg/mL, 1000 mg/5 mL liquid;500 mg tablet); magnesium lactate (84 mg extended release tablet);magnesium oxide (e.g., 140 mg, 600 mg capsules, powder, and/or 200 mg,250 mg, 400 mg, 420 mg, and 500 mg tablets), magnesium sulfate (e.g., 40mg/mL, 80 mg/mL, 125 mg/mL, 500 mg/mL, for injection).

In some embodiments, electrolyte supplementation is administered in anamount sufficient to reduce or delay onset of one or more cardiactoxicities associated with Compound I therapy. In some embodiments, theelectrolyte administration may also reduce one or more of nausea,vomiting, fatigue (lethargy, malaise, asthenia), increased creatinephospho kinase (CPK), hyperuricemia, hypocalcemia, hyperglycemia, fever,gastritis, diarrhea, abdominal pain, dehydration, weight loss,hypophosphatemia, hyponatremia, hypokalemia, hypomagnesemia, syncope,hypoxia, pleural effusion, hypotension, myocardial ischemia, increasedcardiac troponin I, confusion, and/or myelosuppression, and combinationsthereof.

In some embodiments, cardiac toxicities are selected from the groupconsisting of heart-rate corrected QT (QTc) interval prolongation,supraventricular arrhythmias (supraventricular tachycardia (SVT)/atrialfibrillation/flutter), and combinations thereof. In some embodiments,QTc prolongation and/or other electrophysiological changes are reducedto normal values or ranges after electrolyte supplementation.

Unless otherwise defined, all technical and scientific terms used hereinare accorded the meaning commonly known to one of skill in the art. Allpublications, patents, published patent applications, and otherreferences mentioned herein are hereby incorporated by reference intheir entirety. The embodiments of the disclosure should not be deemedto be mutually exclusive and can be combined.

EXAMPLES General Procedures for Characterization of Solid Forms

Provided herein is an assortment of characterizing information todescribe provided forms of Compound I. It should be understood, however,that not all such information is required for one skilled in the art todetermine that such particular form is present in a given composition,but that the determination of a particular form can be achieved usingany portion of the characterizing information that one skilled in theart would recognize as sufficient for establishing the presence of aparticular form, e.g., even a single distinguishing peak can besufficient for one skilled in the art to appreciate that such particularform is present. United States Pharmacopeia provides additional guidancewith respect to characterization of crystalline forms (see, X-RayDiffraction, <941>. United States Pharmacopeia, 31st ed. Rockville, Md.:United States Pharmacopeial Convention; 2008:372-374), which isincorporated herein by reference.

Materials

Solvents were either HPLC grade or ACS grade, unless stated otherwise.Samples were prepared from Compound I Form A solids or from samplesgenerated from these solids. Form designation for the materials wasbased on X-ray powder diffraction (XRPD). Care was taken to protectsamples from light, unless stated otherwise. Prior to characterization,solids were stored as follows: Form A and Form B (may have containedForm A solids as well) under ambient conditions, Form E and Form H overdesiccant in a freezer, Form C in contact with mother liquor in arefrigerator, Form D in contact with mother liquor in a freezer, andForm I in contact with mother liquor under ambient conditions or in afreezer. Due to apparent instability of Form D, all characterizationdata except solution proton nuclear magnetic resonance spectroscopy(¹H-NMR) were collected for Form D on the same day. Although the ¹H-NMRanalysis was not run until a few days later, the solution for theanalysis was prepared on the same day as the rest of thecharacterization.

Instrumental Techniques

Optical Microscopy

Optical microscopy was performed using a Leica MZ12.5 stereomicroscope.Samples were viewed in situ or on a glass slide (sometimes covered inParatone-N oil) with crossed polarizers and a first order redcompensator. Various objectives were used, ranging from 0.8-10×.

X-Ray Powder Diffraction (XRPD) (Inel)

XRPD patterns were collected using an Inel XRG-3000 diffractometerequipped with a curved position sensitive detector with a 2θ range of120°. An incident beam of Cu Kα radiation (40 kV, 30 mA) was used tocollect data in real time at a resolution of 0.03° 2θ. Prior to theanalysis, a silicon standard (NIST SRM 640c) was analyzed to verify theSi 111 peak position. Samples were prepared for analysis by packing theminto thin-walled glass capillaries. Each capillary was mounted onto agoniometer head and rotated during data acquisition. The monochromatorslit was set at 5 mm by 160 μm.

PANalytical Transmission

XRPD patterns were collected using a PANalytical X'Pert Prodiffractometer. An incident beam of Cu Kα radiation was produced usingan Optix long, fine-focus source. An elliptically graded multilayermirror was used to focus the Cu Kα X-rays of the source through thespecimen and onto the detector. Data were collected and analyzed usingX'Pert Pro Data Collector software (v.2.2b). Prior to the analysis, asilicon specimen (NIST SRM 640c) was analyzed to verify the Si 111 peakposition. The specimen was sandwiched between 3 μm thick films, analyzedin transmission geometry, and rotated to optimize orientationstatistics. A beam-stop was used (sometimes with helium gas) to minimizethe background generated by air scattering. Soller slits were used forthe incident and diffracted beams to minimize axial divergence.Diffraction patterns were collected using a scanning position-sensitivedetector (X'Celerator) located 240 mm from the specimen.

PANalytical Reflection

XRPD patterns were collected using a PANalytical X'Pert Prodiffractometer. An incident beam of Cu Kα radiation was produced using aceramic tube with a long, fine-focus source and a nickel filter. Thediffractometer was configured using the symmetric Bragg-Brentanogeometry with a reflection stage and a manually operated spinner. Datawere collected and analyzed using X'Pert Pro Data Collector software (v.2.2b). Prior to the analysis, a silicon specimen (NIST SRM 640c) wasanalyzed to verify the Si 111 peak position. The specimen was preparedas a thin, circular layer centered on a silicon zero-backgroundsubstrate. Anti-scatter slits were used to minimize the backgroundgenerated by air scattering. Soller slits were used for the incident anddiffracted beams to minimize axial divergence. Diffraction patterns werecollected using a scanning position-sensitive detector (X'Celerator)located 240 mm from the specimen.

Peak Identification Process (XRPD)

Peaks within the range of up to about 30° 2θ were selected. Differentrounding algorithms were used to round each peak to the nearest 0.01°2θ, depending upon the instrument used to collect the data and/or theinherent peak resolution. The location of the peaks along the x-axis (°2θ) in both the figures and the tables were automatically determinedusing proprietary software¹ and rounded to two significant figures afterthe decimal point based upon the above criteria. Peak positionvariabilities are given to within ±0.1° 2θ based upon recommendationsoutlined in the USP discussion of variability in x-ray powderdiffraction². For d-space listings, the wavelength used to calculated-spacings was 1.541874 Å, a weighted average of the Cu—K_(α1) andCu—K_(α2) wavelengths. Variability associated with d-spacing estimateswas calculated from the USP recommendation, at each d-spacing, andprovided in the respective data tables. ¹PatternMatch™ 3.0.4.²UnitedStates Pharmacopeia, USP 32, NF 27, Vol. 1, pg. 392, May 1, 2009 <941>X-Ray Diffraction.

For samples with only one XRPD pattern and no other means to evaluatewhether the sample provides a good approximation of the powder average,peak tables contain data identified only as “Prominent Peaks”. Thesepeaks are a subset of the entire observed peak list. Prominent peaks areselected from observed peaks by identifying preferably non-overlapping,low-angle peaks, with strong intensity.

If multiple diffraction patterns are available, then assessments ofparticle statistics (PS) and/or preferred orientation (PO) are possible.Reproducibility among XRPD patterns from multiple samples analyzed on asingle diffractometer indicates that the particle statistics areadequate. Consistency of relative intensity among XRPD patterns frommultiple diffractometers indicates good orientation statistics.Alternatively, the observed XRPD pattern may be compared with acalculated XRPD pattern based upon a single crystal structure, ifavailable. Two-dimensional scattering patterns using area detectors canalso be used to evaluate PS/PO. If the effects of both PS and PO aredetermined to be negligible, then the XRPD pattern is representative ofthe powder average intensity for the sample and prominent peaks may beidentified as “Representative Peaks”.

“Characteristic peaks” are a subset of Representative Peaks and are usedto differentiate one crystalline polymorph from another crystallinepolymorph. Characteristic peaks are determined by evaluating whichrepresentative peaks, if any, are present in one crystalline polymorphof a compound against all other known crystalline polymorphs of thatcompound to within ±0.1 °2θ. Not all crystalline polymorphs of acompound necessarily have at least one characteristic peak.

Differential Scanning Calorimetry (DSC)

DSC was performed using a TA Instruments Q2000 differential scanningcalorimeter. Temperature calibration was performed using NIST traceableindium metal. The sample was placed into an aluminum DSC pan, and theweight was accurately recorded. The pan was covered with a lid, and thelid was crimped. A weighed, crimped aluminum pan was placed on thereference side of the cell. The sample cell was equilibrated at theinitial temperature and heated under a nitrogen purge. Reportedtemperatures are at the transition maxima, unless stated otherwise.

Modulated Differential Scanning Calorimetry (MDSC)

MDSC data were obtained on a TA Instruments Q2000 differential scanningcalorimeter equipped with a refrigerated cooling system (RCS).Temperature calibration was performed using NIST traceable indium metal.The sample was placed into an aluminum DSC pan, and the weight wasaccurately recorded. The pan was covered with a lid perforated with alaser pinhole, and the lid was crimped or crimped thenhermetically-sealed pan. A weighed, crimped aluminum pan was placed onthe reference side of the cell. Data were obtained using a modulationamplitude of ±0.50° C. and a 60 second period with an underlying heatingrate of 2.00° C./minute from −50.00 to 200.00° C. The reported glasstransition temperatures are obtained from the inflection point of thestep change in the reversing heat flow versus temperature curve.

Thermogravimetric Analysis (TGA)

TG analysis was performed using a TA Instruments 2050 thermogravimetricanalyzer. Temperature calibration was performed using nickel andAlumel™. The sample was placed in an aluminum pan and inserted into theTG furnace. In one embodiment, the pan was left open. The sample cellwas equilibrated at the initial temperature and the furnace was heatedunder nitrogen. In another embodiment, the instrument was operated undera flow of helium at 10 and 90 cc/min for the purge and balance,respectively, and the furnace was heated under helium at a rate of 20°C./minute to a final temperature of 250° C.

Infrared Spectroscopy (FT-IR)

In one embodiment, FT-IR spectra for solid forms described herein wereacquired on Magna-IR 860® Fourier transform infrared (FT-IR)spectrophotometer (Thermo Nicolet) equipped with an Ever-Glo mid/far IRsource, an extended range potassium bromide (KBr) beamsplitter, and adeuterated triglycine sulfate (DTGS) detector. Some amorphous solid formFT-IR spectra were acquired using Nexus 670®, equipped in the same wayas described for Magna-IR 860® above. Wavelength verification forMagna-IR 860® and Nexus 670® were performed using NIST SRM 1921b(polystyrene). An attenuated total reflectance (ATR) accessory(Thunderdome™, Thermo Spectra-Tech), with a germanium (Ge) crystal wasused for data acquisition. A background data set was acquired with aclean Ge crystal. A Log 1/R (R=reflectance) spectrum was obtained bytaking a ratio of these two data sets against each other.

In another embodiment, FT-IR spectra were acquired on a Nexus 670®Fourier transform infrared spectrophotometer (Thermo Nicolet) equippedwith an Ever-Glo mid/far IR source, a potassium bromide (KBr)beamsplitter, and a deuterated triglycine sulfate (DTGS) detector.Wavelength verification was performed using NIST SRM 1921b(polystyrene). An attenuated total reflectance (ATR) accessory(Thunderdome™, Thermo Spectra-Tech), with a germanium (Ge) crystal wasused for data acquisition. Each spectrum represents 512 co-added scanscollected at a spectral resolution of 2 cm⁻¹. A background data set wasacquired with a clean Ge crystal. A Log 1/R (R=reflectance) spectrum wasobtained by taking a ratio of these two data sets against each other.

Peak positions were determined using standard spectral software. Peakposition variabilities are given to within ±2 cm⁻¹, based on theobserved sharpness of the peaks picked and acquisition of data using a 1cm⁻¹ data point spacing (2 cm⁻¹ resolution). The accuracy and precisionassociated with any particular measurement reported herein has not beendetermined.

Nuclear Magnetic Resonance (NMR)

Solution proton nuclear magnetic resonance spectra (₁H-NMR) wereacquired with a

Varian UNITYINOVA-400 spectrometer. Samples were prepared as solutionsin deuterated dimethylsulfoxide (DMSO-d₆).

Raman Spectroscopy

Raman spectra were acquired on a FT-Raman 960 spectrometer (ThermoNicolet) equipped with a germanium (Ge) detector. Wavelengthverification was performed using sulfur and cyclohexane. Each sample wasprepared for analysis by placing the sample into a 13 mm diametergold-coated cup and leveling the material. Each spectrum represents 512co-added scans collected at a spectral resolution of 2 cm⁻¹.

Example 1 General Preparation of Compound I

Various preparations and purifications of Compound I were described inU.S. Pat. No. 4,977,138, issued Dec. 11, 1990 and International PCTApplication WO02/20817, filed Aug. 22, 2001, each of which isincorporated herein by reference in their entireties.

In some embodiments, producing, purifying and/or storing Compound I atan apparent pH less than approximately 6.5 and/or at an apparent pH ofabout less than approximately 6.0 has been found to prevent theformation of dimerized, oligomerized or polymerized Compound I, asdescribed in US Patent Application Publication No. US 20090186382, filedDec. 28, 2007, which is incorporated herein by reference. In oneembodiment, one or more of the purification steps are performed at anapparent pH less than 6.5. In another embodiment, one or more of thepurification steps are performed at an apparent pH less than 6.0. Incertain embodiments, one or more purification steps are performed at anapparent pH ranging from 4.0 to 6.0. In certain embodiments, all of thepurification steps are carried out at an apparent pH ranging fromapproximately 4.0 to approximately 6.0. In order to prevent theformation of undesired contaminants, the apparent pH of a solutioncontaining Compound I is not allowed to reach an apparent pH aboveapproximately 7.0, or more preferably above approximately 6.0. Theapparent pH of all purification processes is preferably monitored andsubsequently adjusted, if need be, to an apparent pH below approximately6.0. In certain embodiments, it is maintained within the apparent pHrange of approximately 4.0 to approximately 6.0. The control of apparentpH in purification steps towards the end of the process or steps usingaqueous solutions have been found to be particularly useful indiminishing or eliminating the formation of undesired contaminants. Anyacid or buffer may be used to control pH. In certain embodiments, anorganic acid such as acetic acid or formic acid is used to control pH inone of more of the purification steps. In certain embodiments, aninorganic acid such as phosphoric acid or hydrochloric acid is used.

Any procedure for purifying Compound I, whether from fermentation,semi-synthesis, or total synthesis, can be modified based on the presentdisclosure to prevent the formation of undesired side products bymonitoring apparent pH and reducing the apparent pH, if necessary.

Exemplary data for Compound I in the form of ¹H-NMR in depicted in FIG.1( a) and a molecular structure of Compound I is depicted in FIG. 1( b).The ¹H-NMR depicted in FIG. 1( a) displays chemical shifts andintegration consistent with Compound I, has residual acetone present (atapproximately 2.08 ppm) and the water peak (occurring at 3.33 ppm) hasbeen truncated.

Example 2 Preparation and Characterization of Form C and/or CompositionsContaining Form C

Compound I Form C was prepared via serial seeding of saturated solutionsof romidepsin Form A with solids containing Compound I Form C, with theresulting X-ray powder diffraction (XRPD) pattern of each generatedmaterial exhibiting more reflections present in the Compound I Form Cpattern than the last. The series included three experiments: (a) FirstSeeding Procedure; (b) Second Seeding Procedure; and (c) FinalPreparation of Compound I Form C. An XRPD pattern collected for finalproduct Compound I Form C does not appear to exhibit reflections fromCompound I Form A. The experiments were conducted as follows:

(a) First Seeding Procedure-Preparation of Portion 1 and Portion 2Samples

Compound I Form A (103 mg, 0.2 mmol) and acetone (5 mL) were charged toa glass vial and vortexed for approximately 1 minute, generating a clearsolution. The vial was immersed in a −5° C. bath, as measured by aNIST-traceable thermometer. The sample was left in the bath unstirredfor approximately 26 hours, producing a slight precipitate. Theprecipitate was removed via filtration through a 0.2 μm nylon filterdisc to a clean glass vial, resulting in a clear solution.

While the solution was still cold, cold water (15 mL) was added, withoutagitation. The solution remained clear and cold, with no visibleprecipitate, and the sample was returned to the −5° C. bath. The samplewas left in the bath unstirred for approximately 5 days. After the firstnight, the vial was gently shaken before returning to the bath,resulting in no apparent change in the sample. After the 5 days, solidswere observed on the bottom of the vial.

The supernatant was decanted off and the solids were gently crushed,producing slurry. A portion of the slurry (“portion 1”) was centrifugedin small aliquots at ambient temperature in a 1.0 mm glass capillary,for analysis by X-ray powder diffraction. Centrifugation was done inincrements of several seconds to approximately 10 minutes, with totalcentrifugation more than 20 minutes. X-ray powder diffraction analysisshowed evidence of reflections present in Compound I Form A and CompoundI Form C, suggesting the recovered solids were a mixture of phases.

A second portion (“portion 2”) was left open in a vial at ambienttemperature to partially dry the solids while a capillary was beingprepared. Both capillary and bulk samples were stored in a refrigeratorbefore and after the analysis. The capillary sample was analyzed shortlyafter preparation and the bulk sample was used as seed on the day of itsisolation.

(b) Second Seeding Procedure

Compound I Form A (1.03 g, 1.9 mmol) and acetone (37 mL) were charged toa glass vial and vortexed briefly, generating a clear solution. The vialwas immersed in a −5° C. bath, as measured by a NIST-traceablethermometer. The sample was left in the bath unstirred for approximately1.5 hours, producing a relatively small amount of precipitate. Theprecipitate was removed via cold filtration through a 0.2 μm nylonfilter disc to a clean glass round bottom flask.

The flask contained solids from “portion 2” (the amount approximatelythat of a spatula tip) as seed, to encourage formation of Compound IForm C. No precipitate was apparent but the seed solids remained.Additional solids from “portion 2” (the amount approximately that of aspatula tip) were added. No precipitate was apparent but the seed solidsremained.

Cold water (111 mL) was poured in all at once. After a few minutes,there appeared to be a slight precipitate. The flask was immersed in the−5° C. bath overnight. Only a slight precipitate was observed. Thesample was briefly shaken and returned to the bath for approximately 2hours, resulting in substantial precipitate. Solids were gently scrapeddown from the flask walls.

Targeting solids on the flask bottom, “portion 3” was centrifuged insmall aliquots at ambient temperature in a 1.0 mm glass capillary, foranalysis by X-ray powder diffraction. Centrifugation was done inincrements of several seconds. X-ray powder diffraction analysis showedthe recovered solids to consist mainly of Compound I Form C, andindications of presence of Compound I Form A.

The sample was stored in a refrigerator before and after the analysisbut was not analyzed until the next day. Analysis occurred shortly afterremoval from the refrigerator (“portion 4”). “Portion 4” was left sealedat ambient temperature while the capillary was being prepared, returnedto the −5° C. bath for approximately 3 days and then stored in arefrigerator briefly before being used as seed.

(c) Final Preparation of Form C

Compound I, Form A (1.09 g, 2.0 mmol) and acetone (39 mL) were chargedto a glass vial, vortexed and briefly bath sonicated, generating a clearsolution. The vial was immersed in a −5° C. bath, as measured by aNIST-traceable thermometer. The sample was left in the bath unstirredfor approximately 2.5 hours, producing a relatively small amount ofprecipitate. The solid precipitate was removed via cold filtrationthrough a 0.2 μm nylon filter disc to a clean glass round bottom flask,resulting in a clear solution.

The flask was seeded with slurry from “portion 4” (approximately 1 mL),to encourage formation of Compound I Form C. No precipitate was apparentbut the seed solids remained.

Cold water (400 mL) was poured in all at once. There appeared to be avery slight precipitate and the seed solids persisted. Additional slurryfrom “portion 4” (approximately 1 mL) was added, with the same result,even after briefly swirling the flask. The flask was immersed in the −5°C. bath for approximately 3 days, freezing the solvent.

After leaving the flask in the refrigerator overnight, the solventmelted but solids remained. The flask was swirled and the sample wascentrifuged in 50 mL aliquots at ambient temperature in two plasticcentrifuge tubes simultaneously. Centrifugation was done in incrementsof approximately 5 to 10 minutes, minimizing warming of the sample andensuring clear supernatant was generated. The resulting supernatantswere decanted off to a clean HDPE bottle. The final flask aliquotincluded rinsing once with liquid from the bottle (several mL) torecover additional solids from the flask walls. These solids did notappear to be new precipitate but collected on the walls when pouringsample from the flask into the tubes. Little residual sample was presentin the flask and this residual was not recovered. After the flask samplewas exhausted, the centrifuged samples were recovered to one tube,rinsing the other tube twice with liquid from the bottle (approximately15 mL per rinse). The final supernatant was left with the solids. Thetubes, flask and bottle were stored in a refrigerator when not beingmanipulated. This included overnight storage since the centrifugationwas completed over two days and solids were not isolated until the dayafter centrifugation.

Targeting the solids on the tube bottom, a portion of final productCompound I Form C was centrifuged in small aliquots at ambienttemperature in a 1.0 mm glass capillary, for immediate analysis by X-raypowder diffraction. Centrifugation was done in increments of severalseconds.

Exemplary data for Compound I Form C in the form of X-ray diffractionpatterns (XRPD), differential scanning calorimeter thermograms (DSC),thermogravimetric analysis thermograms (TGA), infrared spectrums(FT-IR), and single crystal structure data (e.g., ORTEP drawings,packing diagrams, positional parameters, bond distances and bond angles)are depicted in FIGS. 1( c) through 1(q), supra. A summary of exemplarydata presented in FIGS. 1( c) through 1(q) is as follows.

Form C is a crystalline non-stoichiometric hydrate of Compound I, asdetermined from single crystal data (see FIGS. 1( i) through 1(q)). Thecrystal structure contains one fully occupied water molecule and asecond water site with a refined occupancy of approximately 73%. Thecharacterization of Compound I, Form C is summarized in Table 3.

TABLE 3 Characterization of Compound I Form C Analysis Result FigureReferences XRPD Form C 1(c), 1(d), 1(i), 1(j) DSC 96.6° C. (broad endo,min) 1(e) 139.6° C. (broad endo, min) 177.2° C. (broad exo, max) 257.1°C. (endo, min) followed by decomp. TGA 5.3 wt % loss to 103° C. 1(f)FT-IR reference spectrum 1(g), 1(h) Single Crystal Form C 1(i)-1(q)X-ray (non-stoichiometric hydrate, ~1.7 (non-GMP) waters)

A comparison of the XRPD pattern final product for Compound I Form C(see FIGS. 1( c) and 1(d)); and the calculated pattern collected atsubambient temperature (see FIGS. 1( i) and 1(j)) from the structure ofCompound I, Form C, suggests that the XRPD patterns represent a singlephase and that none of the observed reflections are attributed toCompound I Form A. The single crystal data were collected at cryogenictemperature, so minor, uneven shifting of 2θ peak positions due totemperature effects was observed.

The differential scanning calorimetry (DSC) thermogram for Compound I,Form C (see FIG. 1( e)) exhibits broad endothermic events atapproximately 97° C. and 140° C. (min), ascribed to loss of solvent,based on the 5.3% weight loss observed in the thermogravimetric analysis(TGA) thermogram (see FIG. 1( f)). This weight loss corresponds toapproximately 1.7 moles of water, which is similar to the resultobtained from the single crystal data. However, the loss may includeacetone, since the sample was crystallized from an acetone/watermixture. The DSC thermogram also exhibits an endotherm at approximately257° C. (min) (see FIG. 1( e)). This endotherm is believed to correspondto the melt of Compound I Form A and apparent desolvation of solids. Aminor exothermic event was observed at approximately 177° C. (see FIG.1( e)). Based on the apparent melting temperature, this appears torepresent recrystallization to Compound I Form A. The final weight lossfrom TGA suggests that decomposition is concurrent with the apparentmelt observed by DSC, as it was for Compound I Form A. Solids wereair-dried in a laboratory fume hood at ambient temperature forapproximately 2.5 hours to remove residual solvent before the analyses,in order to obtain representative thermal data for Compound I, Form C.

One skilled in the art will be able to readily ascertain from the datapresented that Form C may be isostructural with the methanol solvatereported in Shigematsu et al., The Journal of Antibiotics, Vol. 47, No.3, “FR901228, A Novel Antitumor Bicyclic Depsipeptide Produced byChromobacterium violaceum No. 968, pp. 311-314 (March 1994).

Example 3 Preparation and Characterization of Form D and/or CompositionsContaining Compound I Form D

Compound I Form A (1.20 g, 2.2 mmol) and acetone (38 mL) were charged toa glass Erlenmeyer flask, shaken, swirled, and bath sonicated for a fewminutes, dissolving most of the solids. Undissolved solids were removedvia filtration through a 0.2 μm nylon filter disc to a clean glassErlenmeyer flask, resulting in a clear solution. Hexanes (152 mL) wasadded, which precipitated solids immediately, without agitation. Theflask was left in a freezer overnight, allowing the solids to settle tothe bottom of the flask. The clear supernatant was decanted off andaliquots of solid were removed for immediate X-ray powder diffractionanalysis. The analysis showed that the solids consisted of Compound IForm D. Solids were recovered from the analysis sample for thermal andspectroscopic analyses. Unused material was stored in the freezer.

Exemplary data for Compound I Form D in the form of an XRPD, a DSC, aTGA and an FT-IR are depicted in FIGS. 2( a) through 2(f), supra. Asummary of exemplary data presented in FIGS. 2( a) through 2(f) is asfollows. As described in Example 8, one skilled in the art will be ableto readily ascertain from the data presented herein that Compound I FormD may be isostructural with MEK solvate (Compound I Form J).

Form D is an unstable crystalline acetone solvate of Compound I thatconverts to Form A under ambient conditions. A crystal prepared fromcold acetone solution was indexed. The indexing solution was determinedto be an orthorhombic unit cell with the following cell parameters andcalculated volume: a=9.093, b=15.581, c=23.141 Å, V=3278.57(9) Å³. Theformula weight was determined to be 598.81 g/mol. The cell parametersare similar to the cell obtained from the Compound I Form J crystalstructure. The similarity between the two unit cells and XRPD patternsof Compound I Form D and Compound I Form J suggest the two samples arerelated crystal forms. Since Compound I Form J was determined to be amono methyl ethyl ketone solvate of Compound I, it is likely that Form Dis also a mono solvate of Compound I. Characterization of Compound IForm D is summarized in Table 4.

TABLE 4 Characterization of Compound I Form D Analysis Result FigureReferences XRPD Form D 2(a), 2(b) DSC 91.4° C. (exo, max) 2(c) 260.6° C.(endo, max) followed by decomp TGA 10.9 wt % loss to 63° C. 2(d) FT-IRreference spectrum 2(e), 2(f)

An experimental Compound I Form D pattern is provided in FIG. 2( a) withan accompanying line list in FIG. 2( b). The pattern is consistent witha pattern for Compound I Form D and similar to a pattern for Compound IForm J as observed in the XRPD overlay presented in FIG. 6( a). Thishigh resolution pattern of FIG. 2( a) was collected after storage of thematerial in a freezer and displayed presence of Compound I Form D andCompound I Form A, suggesting a mixture of phases, so the patterngenerated from the material after storage in the freezer was used togenerate a corresponding peak list for Compound I Form D (see FIG. 2(b)).

An FT-IR spectrum of Compound I Form D and accompanying peak list isprovided as FIG. 2( e) and FIG. 2( f). To avoid the potential for formconversion from solvent loss, the solids for the FT-IR data werecollected immediately upon removal from the freezer.

The TGA thermogram for Compound I Form D (see FIG. 2( d)) exhibits aweight loss of approximately 10.9% and the DSC thermogram (see FIG. 2(c)) exhibits a small exothermic event at approximately 91° C. Theseevents appear to be mainly related to desolvation and recrystallizationto Compound I Form A, respectively, based on the instability of CompoundI Form D and tendency for conversion to Compound I Form A. The weightloss observed by TGA corresponds to slightly more than a mole ofacetone. To avoid the potential for Form conversion from solvent loss,the solids were analyzed immediately upon removal from the freezer.Since no weight loss was observed prior to the start of the analysis,the weight loss observed is attributed to solvent loss from the crystallattice, also suggesting Compound I Form D is an acetone solvate. TheDSC thermogram also exhibits an endotherm at approximately 261° C.(min). The endotherm is believed to correspond to a melt of Compound IForm A and apparent desolvation of the solids. Final weight loss fromTGA suggests that decomposition is concurrent with apparent meltobserved by DSC, as it was for Compound I Form A.

Example 4 Preparation and Characterization of Form E and/or CompositionsContaining Form E

Compound I Form A (2.75 g, 5.1 mmol) and solution containing a mixtureof t-butanol and water [60:40 (v/v)] (31 mL) were charged to a 50 mLErlenmeyer flask. Solids remained. The sample was stirred overnight atambient temperature and the resulting solids were collected by vacuumfiltration. The recovered solids were transferred to weigh paper anddried under ambient conditions for approximately 2 hours. The driedsolids were transferred to a glass vial and stored under ambientconditions. X-ray powder diffraction analysis showed the solids toconsist of Compound I Form E. Solid recovery was 2.79 g (89%).

Exemplary data for Compound I Form E in the form of an XRPD, a DSC, aTGA an FT-IR, a Raman spectrum and single crystal structure data (e.g.,ORTEP drawings, packing diagrams, positional parameters, bond distancesand bond angles) are depicted in FIGS. 3( a) through 3(p), supra. Asummary of exemplary data presented in FIGS. 3( a) through 3(p) is asfollows. One skilled in the art will be able to readily ascertain fromthe data presented herein that Compound I, Form E may be isostructuralwith Compound I, Form H (see Example 6).

Compound I Form E is a crystalline mono-tert-butanol solvate of CompoundI, as determined from single crystal data (see FIGS. 3( h) through3(p)). The characterization of Compound I Form E is summarized in Table5.

TABLE 5 Characterization of Compound I Form E Analysis Result FigureReferences XRPD Form E 3(a), 3(b), 3(h), 3(i) DSC 158.1° C. (broad endo,peak) 3(c) 255.3° C. (endo, max) followed by apparent decomp. TGA 10.9wt % loss to 200° C. 3(d) Single Crystal X-ray Form E 3(h)-3(p)(non-GMP) (mono tert-butanol solvate) FT-IR reference spectrum 3(e),3(f) Raman reference spectrum 3(g)

A comparison of the experimental (see FIGS. 3( a) and 3(b)) andcalculated (see FIGS. 3( h) and 3(i)) XRPD patterns and accompanyingpeak lists of Compound I Form E are provided. The single crystal datawere collected at cryogenic temperature, so minor uneven shifting of 2θpeak positions due to temperature effects was observed. FT-IR with anaccompanying peak list (see FIGS. 3( e) and 3(f)) and FT-Raman spectra(see FIG. 3( g)) of Compound I Form E are provided.

The DSC thermogram for Compound I Form E (see FIG. 3( c)) exhibits anendothermic event at approximately 158° C. (min), ascribed todesolvation, based on the TGA thermogram (see FIG. 3( d)), and indicatedby hot stage microscopy as partial loss of birefringence atapproximately 157° C. Hot stage microscopy showed the specimen to meltat approximately 243° C., as indicated by an endotherm in the DSC atapproximately 255° C. (min). Based on the melting temperature, it isbelieved that the sample desolvated to Compound I Form A prior to melt.The final weight loss from TGA suggests that decomposition is concurrentwith the melt observed by hot stage microscopy, as it was for Compound IForm A.

Example 5 Preparation and Characterization of Form F and/or CompositionsContaining Form F

In one embodiment, compound I Form A (105.9 mg, 0.2 mmol) and chloroform(4 mL) were charged to a glass vial and bath conicated for approximately1 minute, generating a clear solution, with a few undissolved particles.Additional Compound I Form A (281.7 mg, 0.5 mmol) was added. Theresulting slurry was agitated on a rotating wheel under ambientconditions for ˜12 hours. The sample was removed from the wheel and theremaining solids floated to the top of the solution. The solution wasdrawn off with a pipet and a portion was filtered through a 0.2 μm nylonfilter disc to a clean glass vial. The vial was left open to evaporatein an ambient laboratory fume hood. The recovered solids were analyzedby X-ray powder diffraction (XRPD) and consist of Compound I Form F.

In another embodiment, Compound I Form A (740 mg, 1.4 mmol) andchloroform (30 mL) were charged to a glass vial and bath sonicated for afew minutes, producing a clear solution. Compound I Form A (750 mg, 1.4mmol) was added to ensure excess solids for slurry. The resulting samplewas agitated for approximately 4 days on a rotating wheel. Remainingsolids floated to the top upon standing, generating a clear solution atthe bottom of the vial. Approximately ¼ of the solution was drawn off toa clean glass vial and solids were precipitated via slow evaporation ofthe solvent (vial covered with perforated aluminum foil) in a laboratoryfume hood. After approximately 2 days, no solvent was apparent. Thesolids consisting of Compound I Form F were left in a sealed vial atambient temperature for approximately 1 day, and then stored in afreezer.

Exemplary data for Compound I Form F in the form of an XRPD and an FT-IRare depicted in FIGS. 9( a) through 9(l), supra.

Compound I Form F is a crystalline chloroform solvate of Compound I. Thecharacterization of Compound I Form F is summarized in Table 6.

TABLE 6 Characterization of Compound I Form F Analysis Result FigureReferences XRPD Form F 9(a)-9(f), 9(h), 9(i) DSC 83.6° C. (minor endo)9(j) 97.3° C. (endo) 256.4° C. (endo) FT-IR reference spectrum 9(g),9(h) TGA Form F 9(k)

Example 6 Preparation and Characterization of Form H and/or CompositionsContaining Form H

Compound I Form A (500 mg, 0.9 mmol) and chloroform (5 mL) were chargedto a glass vial and bath sonicated for approximately 20 minutes, andgenerated a clear solution. Gentle shaking produced solid precipitate.The resulting mixture was agitated on a rotating wheel overnight atambient temperature. Solids were floating on top of the liquid, so theliquid was drawn off with a pipette. Approximately ⅓ of the solids weredried via rotary evaporation over approximately 15 minutes, utilizing awater bath. The temperature range of the bath during evaporation was 57to 64° C., as measured by a NIST-traceable thermometer. The recoveredsolids were stored under ambient conditions until analyzed by XRPD. Theanalysis showed the solids to consist of Compound I Form H. After XRPDanalysis, the sample was stored in a freezer with desiccant. Solidrecovery was 178 mg.

Exemplary data for Compound I Form H in the form of an XRPD, a DSC, aTGA, and an FT-IR are depicted in FIGS. 4( a) through 4(f), supra. Asummary of exemplary data presented in FIGS. 4( a) through 4(f) is asfollows. One skilled in the art will be able to readily ascertain fromthe data presented herein that Compound I, Form H may be isostructuralwith Compound I, Form E (see Example 4).

Compound I Form H is a crystalline chloroform solvate of Compound I.Characterization of Compound I Form H is summarized in Table 7.

TABLE 7 Characterization of Compound I Form H Analysis Result FigureReferences XRPD Form H 4(a), 4(b) DSC 96.3° C. (broad endo, peak) 4(c)256.7° C. (endo, max) followed by apparent decomp TGA 10.1 wt % loss to150° C. 4(d) FT-IR reference spectrum 4(e), 4(f)

A high resolution XRPD pattern of Compound I Form H and an accompanyingline list is provided in FIGS. 4( a) and 4(b). An FT-IR spectrum ofCompound I Form H and an accompanying line list is provided in FIGS. 4(e) and 4(f).

Examination of Compound I Form H XRPD pattern displays reflections fromboth Compound I Form H and Compound I Form A patterns, suggesting thespecimen examined was a mixture. The XRPD pattern generated usingCompound I Form H appears to be consistent with the Form H portion ofthe pattern.

The DSC thermogram for Compound I Form H (see FIG. 4( c)) exhibits anendothermic event at approximately 96° C. (min). This event appears tobe mainly related to desolvation, based on the weight loss ofapproximately 10.1% observed in the TGA thermogram for Compound I Form H(see FIG. 4( d)). This corresponds to more than 0.5 moles of chloroform.To avoid the potential for Form conversion from solvent loss, the solidswere analyzed immediately upon removal from the freezer. Since no weightloss was observed prior to the start of the analysis, the weight lossobserved is attributed to solvent loss from the crystal lattice,suggesting Compound I Form H is a solvate. The DSC thermogram (see FIG.4( c)) also exhibits an endotherm at approximately 256° C. (min). Theendotherm is believed to correspond to the melt of Compound I Form A andapparent desolvation of solids. Final weight loss from TGA (see FIG. 4(d)) suggests that decomposition is concurrent with the apparent meltobserved by DSC, as it was for Compound I Form A.

Example 7 Preparation and Characterization of Form I and/or CompositionsContaining Form I

In one embodiment, compound I Form A (500 mg, 0.9 mmol) and chloroform(5 mL) were charged to a glass vial and bath sonicated for approximately20 minutes, and generated a clear solution. Gentle shaking producedsolid precipitate. The resulting mixture was agitated on a rotatingwheel at ambient temperature for less than an hour and a portion of thesolids was recovered for X-ray powder diffraction (XRPD) via filtrationwith a 0.22 μm nylon filter in a Swinnex Millipore filter body. Thefilter cake was not washed and the solids appeared dry upon recovery.The solids were gently crushed prior to XRPD analysis. The analysisshowed presence of Compound I Form I and Compound I Form H, suggestingthe recovered solids were a mixture of phases.

The remaining sample was returned to the wheel to slurry overnight. Thesolids were floating on top of the liquid, so the liquid was drawn offwith a pipette. The remaining solids were stored in a sealed vial overdesiccant in a freezer. An attempt to collect a high resolution XRPDdata indicated the solids converted to Compound I Form H prior toanalysis.

In another embodiment, compound I Form A, (517 mg, 1.0 mmol) andchloroform (5 mL) were charged to a glass vial and bath sonicated forapproximately 20 minutes, generating a clear solution, with a trace ofsolid. The resulting mixture was agitated on a rotating wheel forapproximately 1 month at ambient temperature. The solids were stored inthe mother liquor in a refrigerator. A portion of the solids (“portion1”) was recovered for X-ray powder diffraction (XRPD) via filtrationwith a 0.22 μm nylon filter in a Swinnex Millipore filter body. Thefilter cake was not washed and the solids appeared dry upon recovery.The solids were gently crushed prior to XRPD analysis. The analysisshowed that the solids consisted of Compound I Form I. Another portion(“portion 2”) of the solids was recovered for solution proton nuclearmagnetic resonance spectroscopy (¹H-NMR) by pipetting to a clean glassvial and decanting off the liquid. The XRPD and ¹H-NMR samples werestored at ambient temperature in sealed vials prior to analysis.

In yet, another embodiment, Compound I Form A (˜180 mg, 0.3 mmol) wascharged to a glass vial. The vial was left uncapped in a glass jarcontaining chloroform (˜10 mL), for vapor stress of the solids. Thesolids were stressed for approximately 7 days before transfer to afreezer, where they remained under chloroform vapor.

Exemplary data for Compound I Form I in the form of XRPDs, a DSC, a TGA,an FT-IR, and single crystal structure data (e.g., ORTEP drawings,packing diagrams, positional parameters, bond distances and bond angles)are depicted in FIGS. 5( a) through 5(y), supra. A summary of exemplarydata presented in FIGS. 5( a) through 5(y) is as follows.

Compound I Form I is a crystalline chloroform solvate of Compound I thatconverts to Form H under ambient conditions. The structure was solvedfor a crystal prepared from chloroform slurry. Based on Compound I FormI XRPD pattern from a sub sample of the bulk solids, it is believed thecrystal was of Compound I Form I. The single crystal data (see FIGS. 5(g) through 5(o)) indicate chloroform solvate, the structure consistingof layers of Compound I molecules separated by residual electron densitybelieved to be free chloroform and pockets containing refined chloroformmolecules.

The experimental data for Compound I Form I is provided in FIGS. 5( a)to 5(y). Characterization of Compound I Form I is summarized in Table 8.

TABLE 8 Characterization of Compound I Form I Analysis Result FigureReferences XRPD Form I 5(a), 5(b), 5(p)-5(r) DSC 73.8° C. (broad endo,max) 5(c) 100.2° C.(endo, min) 257.8° C. (endo, min) followed by decompTGA 33.0 wt % loss 19 to 102° C. 5(d), 5(x) FT-IR reference spectrum5(e), 5(f), 5(s), 5(t) Single Crystal Form I 5(g)-5(o) X-ray (chloroformsolvate) (non-GMP)

The initial precipitate and the isolated solids from slurry inchloroform both exhibited an XRPD pattern consistent with Compound IForm I. The high resolution XRPD pattern collected on a sample of bulksolids appears to be Compound I Form H. Because Compound I Form H wasprepared by drying solids exhibiting XRPD pattern for Compound I Form I,it is possible that the sample converted to Compound I Form I duringdata collection. In contrast, the initial XRPD data for Compound I FormI solids were collected on solids in a glass capillary, thus retardingthe drying of the solids.

The DSC thermogram for Compound I Form I (see FIG. 5( c)) exhibits abroad endothermic event at approximately 74° C. and an endothermic eventat approximately 100° C. (min). These events appear to be mainly relatedto desolvation, based on the weight loss of approximately 33% from 19 to102° C. observed in the TGA thermogram (see FIG. 5( d)). Thiscorresponds to more than 2 moles of chloroform. The TGA thermogram alsoexhibits weight loss prior to 19° C., which is likely due to residualchloroform; however, there appears to be a clear transition into themain weight loss. The DSC thermogram (see FIG. 5( c)) also exhibits anendotherm at approximately 258° C. (min). The endotherm is believed tocorrespond to the melt of Compound I Form A and the apparent desolvationof the solids. The final weight loss from TGA suggests thatdecomposition is concurrent with the apparent melt observed by DSC, asit was for Compound I Form A. In an attempt to avoid the potential forform conversion from solvent loss, the solids were analyzed immediatelyupon removal from the freezer.

An FT-IR spectrum of Compound I Form I (see FIGS. 5( e) and 5(s)) isprovided. To avoid potential for Form conversion from solvent loss,solids were analyzed immediately upon removal from the freezer.

Example 8 Preparation and Characterization of Form J and/or CompositionsContaining Form J

In one embodiment, Compound I (56.4 mg) Form J was dissolved in methylethyl ketone (4.5 mL). The solution was filtered through a 0.2-μm nylonfilter. The sample was placed in a vial capped with perforated aluminumfoil (single pinhole) in a laboratory fume hood and allowed to evaporateto dryness under ambient conditions. The sample was stored under ambientconditions until indexed by single crystal X-ray. Crystallization may beperformed using methods known to one of skill in the art.

In another embodiment, Compound I Form A (Sandoz lot 49800203, 1.03 g,1.9 mmol) and methyl ethyl ketone (80 mL) were charged to an Erlenmeyerflask, briefly swirled and bath sonicated for a few minutes, producing aclear solution. Approximately half of the solution was filtered througha 0.2 μm nylon filter to a clean glass vial. The vial was capped andplaced into a freezer, in order to precipitate solids from the solution.After approximately 5 days, the sample was removed from the freezer andthe precipitated solids were isolated by decanting off the clearsupernatant. The solids were stored wet with solvent in a freezer.

Exemplary data for Compound I Form J in the form of single crystalstructure data (e.g., ORTEP drawings, packing diagrams, positionalparameters, bond distances and bond angles) are depicted in FIGS. 6( a)through 6(j), supra. A summary of exemplary data presented in FIGS. 6(a) through 6(j) is as follows.

The single crystal structure of Compound I Form J confirmed themolecular structure and the contents of the unit cell. The samplecrystallized in the chiral orthorhombic space group P2₁2₁2₁ and wasdetermined to be a methyl ethyl ketone (MEK) solvate of Compound I. Thestructure of Compound I Form J (MEK solvate) consists of layers ofCompound I molecules hydrogen bonded to neighboring Compound I moleculerunning perpendicular to the crystallographic c axis. The reflections inthe experimental pattern of the acetone solvate (Compound I Form D) arerepresented in the calculated XRPD pattern of the MEK solvate (CompoundI Form J), suggesting that the two forms may be isostructural (seeExample 3).

Compound I Form J is a crystalline methyl ethyl ketone solvate ofCompound I. The experimental data for Compound I Form J is provided inFIGS. 6( a) to 6(s). The characterization of Compound I Form J issummarized in Table 9.

TABLE 9 Characterization of Compound I Form J Analysis Result FigureReferences XRPD Form J 6(a) 6(m) DSC 130.3° C. (endo) 6(q) 260.0° C.(endo) FT-IR reference spectrum 6(n), 6(o) TGA Form J 6(r)

Example 9 Preparation and Characterization of Amorphous Compound Iand/or Compositions Containing Amorphous Compound I

Preparation from 9:1 Dioxane/Water

Compound I (1.0652 g) was dissolved in 9:1 dioxane/water (10 mL). Thesolution was filtered through a 0.2-μm nylon filter, and frozen in a 300mL round-bottom flask immersed in a bath of dry ice and isopropanol. Theflask containing the frozen sample was attached to a lyophilizer anddried for approximately 4 days. After drying, the solids were isolatedand stored in the freezer over desiccant until used.

Preparation by a Rotary Evaporator

Compound I (133.4 mg) was dissolved in dichloromethane (1.5 mL). Thesolution was filtered through a 0.2-μm nylon filter. The sample vial wasplaced on the rotary evaporator and immersed in a water bath at ambienttemperature. The solvent was rapidly evaporated to dryness under vacuum.The solids were then stored in the freezer over desiccant until used.

Preparation by Fast Evaporation

Compound I (24.7 mg) was dissolved in a binary solvent mixture of water(1.5 mL) and dichloromethane (0.5 mL). The solution was filtered througha 0.2-μm nylon filter. The sample was placed uncapped in a laboratoryfume hood and allowed to evaporate to dryness under ambient conditions.The solids were stored under ambient conditions until used.

Exemplary data for amorphous Compound I in the form of XRPD's, modulatedDSC thermogram, TGA, FT-IR, FT-Raman spectroscopy and ¹H NMR aredepicted in FIGS. 7( a) through 7(f), supra. A summary of exemplary data(e.g., a summary of XRPD results in Table 10) are presented foramorphous Compound I below.

A high resolution XRPD pattern of amorphous Compound I is provided inFIG. 7( a). The modulated DSC thermogram for amorphous Compound I (seeFIG. 7( b)) exhibits a glass transition temperature at approximately 91°C. Weight loss of approximately 3.5% was observed in the TGA thermogram(see FIG. 7( c)). An FT-IR spectrum of amorphous Compound I (see FIGS.7( d) and 7(e)) and an FT-Raman spectrum (see FIG. 7( f)) are alsoprovided.

TABLE 10 Preparation of X-ray Amorphous Compound I and/or CompositionsContaining Amorphous Compound I Conditions Description ^(a) XRPD Resultrotary evaporation in dichloromethane white solids, chunk, no B x-rayamorphous + (concentration: 268 mg/mL) Form A rotary evaporation indichloromethane white solids, chunk, no B x-ray amorphous(concentration: 89 mg/mL) fast evaporation (FE) in dichloromethane whitesolids, irregular, B/E not analyzed (concentration: 102 mg/mL) freezedrying in dioxane/water (9:1), 2 white solids, chunk, no B x-rayamorphous days freeze drying in dioxane/water (9:1), 2 white solids,chunk, partial x-ray amorphous + days, ~2 g scale-up B Form A + PatternK + peaks freeze drying in dioxane/water (9:1), 2 white solids, chunk,no B x-ray amorphous + days, ~2 g scale-up Form K + peaks freeze dryingin dioxane/water (9:1), 4 white solids, chunk, no B x-ray amorphousdays, ~1 g scale-up freeze drying in dioxane/water (9:1), 4 whitesolids, chunk, no B x-ray amorphous days, ~1 g scale-up ^(a) B =birefringence, E = extinction.

Example 10 Preparation and Characterization of Compound I, Form K

In one embodiment, Compound I Form A (410 mg, 0.8 mmol) and nitromethane(20 mL) were charged to a glass vial and bath sonicated for severalminutes, producing a clear solution. The solution was filtered through a0.2 μm nylon filter to a clean glass vial and allowed to evaporateslowly (vial covered with perforated aluminum foil) in a laboratory fumehood. After approximately 12 days, the sample was split intoapproximately four equal portions to speed up the evaporation. Thesample was continued as a slow evaporation for an additional 7 days. Twoof the four vials were uncapped (fast evaporation) and allowed toevaporate overnight. The next day, a small amount of solvent was visiblein only one of the samples. After the majority of the solvent wasremoved by decantation, the precipitated solids from the other threesamples were pooled into the original sample. The recombined solids werestored in a sealed vial in a freezer.

Slow Evaporation (SE)

In another embodiment, solutions were prepared in various solvents atambient temperature and passed through a 0.2-μm nylon filter into aglass vial. The filtered solution was allowed to evaporate at ambient ina vial covered with aluminum foil perforated with one or more pinholes.Any solids formed were isolated and analyzed. From nitromethane by slowevaporation, solids obtained display an XRPD pattern for Compound I,Form K (FIG. 8( a).

Vapor Diffusion

In yet another embodiment, solutions were prepared with various solventsat ambient temperature and passed through a 0.2-μm nylon filter into aglass vial. This filled vial was placed in a glass vial containing anantisolvent and capped. In general, the anti-solvent is miscible withand, typically, more volatile than the solvent. The experiment was leftundisturbed at ambient temperature. Any solids formed were isolated andanalyzed.

Two scale-up lyophilization attempts (approx. 2-g scale usingdioxane/water 9:1 v/v) were performed. The first attempt generated adisordered crystalline material with evidence of peaks also found inForm A and Form K as determined by visual comparison of XRPD. The secondattempt generated a disordered crystalline material with evidence ofpeaks also found in Form K by visual comparison.

Compound I Form K is a crystalline nitromethane solvate of Compound I.The experimental data for Compound I Form K is provided in FIGS. 8( a)to 8(l). The characterization of Compound I Form K is summarized inTable 11.

TABLE 11 Characterization of Compound I Form K Analysis Result FigureReferences XRPD Form K 8(a)-8(e), DSC 168.2° C. (endo) 8(i) 259.2° C.(endo) FT-IR reference spectrum 8(f), 8(g) TGA Form K 8(k)

Example 11 Preparation and Characterization of Compound I, Form L

Compound I, Form A (910 mg, 1.7 mmol) and acetone (48 mL) were chargedto a glass beaker and stirred for several minutes, producing a clearsolution. The solution was filtered through a 0.2 μm nylon filter to aclean glass beaker and the beaker was left uncovered in a glass jarcontaining methanol (˜50 mL), in order to precipitate solids from thesolution via vapor diffusion. After approximately 12 days, theprecipitated solids were isolated by decanting off the clearsupernatant. The solids were transferred to a clean glass vial andstored under methanol vapor in a freezer.

Compound I Form L is a crystalline methanole solvate of Compound I. Theexperimental data for Compound I Form L is provided in FIGS. 10( a) to10(i). The characterization of Compound I Form L is summarized in Table12.

TABLE 12 Characterization of Compound I Form L Analysis Result FigureReferences XRPD Form L 10(a)-10(c) DSC 168.2° C. (endo) 10(g) 259.2° C.(endo) FT-IR reference spectrum 10(d), 10(e) TGA Form L 10(h)

Example 12 Preparation and Characterization of Compound I, Form N

In one embodiment, Compound I, Form N was vacuum dried at ambienttemperature for approximately 5 hours, at approximately 50 mTorr, losingapproximately 12.4% of the initial weight. The resulting solids werecharacterized by proton NMR spectroscopy. The spectrum showed that thesolids contained approximately 1/3 mole nitromethane. Subsequently, thedried sample was characterized by DSC. The observed results are subjectto the conditions used at the time of analysis. The DSC data collectedin a crimped pan, exhibits a minor endothermic event at approximately150° C., which may be related to volatiles loss on heating, and anintense endotherm at approximately 256° C. (onset). The remaining solids(43 mg) were dried for approximately 22 hours in a vacuum oven atapproximately 42° C., at approximately 20 mTorr. The weight loss was notdetermined but the dried solids were characterized by XRPD. Theresulting pattern contains XRPD peaks of Form N but exhibits additionalunknown peaks, suggesting conversion had occurred. Therefore, subsequentsolvent removal experiments were carried out at ambient temperature.

Form A (1.6 g, estimated) was slurried in nitromethane (9 mL) forapproximately 5 days. Solids were recovered via vacuum filtration andwashed with nitromethane (2×1 mL). The solids were left on the filterunder vacuum for several minutes. Approximately 1.3 g of solid wererecovered. The solids exhibited a mixture of rectangular plates andprisms by polarized light microscopy. The resulting high-resolution XRPDpattern was consistent with Form N.

Two ambient-temperature vacuum drying experiments were carried out in anattempt to remove the nitromethane from Form N. In one embodiment, 94.0mg of solid were dried for approximately 16.5 hours, at approximately 20mTorr, losing approximately 0.7% of the initial weight. In anotherembodiment, 308.3 mg of solid were dried for approximately 5 days, atapproximately 5 mTorr, gaining approximately 0.7% of the initial weight(approximately 0.1% gain from 3 to 5 days). There appeared to be nochange in the solids by polarized light microscopy and both samplesexhibited Form N by high-resolution XRPD analysis. In one embodiment,the patterns exhibit a weak unknown peak at approximately 9.1 °2θ, whichis more pronounced for the 5-day sample than the 16.5-hour sample. Bothsamples contained approximately 1/3 mole of nitromethane by proton NMRspectroscopy.

In one embodiment, the sample was characterized by DSC and TGA in anopen pan configuration to ensure that solvent could freely leave duringanalysis. The observed results are subject to the conditions used at thetime of analysis. The resulting DSC thermogram exhibits a broadendothermic event at approximately 161° C., with a shoulder atapproximately 148° C. This event appears to be concurrent with theweight loss of approximately 4.9% from 130-160° C. observed in the TGAthermogram, which correlates to approximately 1/2 mole of nitromethane,assuming the weight loss is attributed only to solvent loss. Thethermogram exhibits an endotherm at approximately 256-259° C. (onset).The final weight loss from TGA suggests that decomposition is concurrentwith this endotherm.

In one embodiment, in order to remove the nitromethane from Form Nsample, 152.4 mg of solid were slurried in acetonitrile (1 mL) forapproximately 1 hour. Solids were recovered via vacuum filtration,washing with acetonitrile (4×1 mL). The solids were left on the filterunder vacuum for several minutes to dry the solids. 97.7 mg of solidwere recovered. In another embodiment, 309.5 mg of solid were slurriedin water (4 mL) for approximately 24.5 hours. Solids were recovered viavacuum filtration, washing with water (2×1 mL). The solids were left onthe filter under vacuum for approximately 1.5 hours to dry the solids.270.4 mg of solid were recovered. There was no change in the solids bypolarized light microscopy; however, by XRPD analysis, pattern Tresulted from acetonitrile and a mixture of Forms C and A resulted fromwater. The high-resolution XRPD pattern for the solids from water slurryexhibited additional peaks present in the Form N XRPD pattern,suggesting incomplete conversion.

In addition, the proton NMR data for Form N suggests the materialcontains approximately 1/3 mole of nitromethane. The space group of theForm N solution (P2₁2₁2) can only exhibit less than one molecule ofsolvent in the asymmetric unit if the solvent position is partiallyoccupied, i.e. some of the asymmetric units contain solvent moleculesand others do not.

The experimental data for Compound I Form N is provided in FIGS. 11( a)to 11(c). The characterization of Compound I Form N is summarized inTable 13.

TABLE 13 Characterization of Compound I Form N Analysis Result FigureReferences XRPD Form N 11(a) DSC 150.0° C. (event) 11(b) 259.2° C.(endo) TGA Form N 11 (c)

Characterization of solids from Compound I Form N preparation issummarized in Table 14.

TABLE 14 Characterization of Solids from Romidepsin Form NPreparation/Solvent Removal Attempts Starting Material (XRPD Result)Conditions Analysis Result (Form N + 10.3 mg, DSC (sample preparationpeaks) ^(a) heated to 180° C. (crimped) analysis) by DSC XRPD Form A + 2weak peaks of Form N (Form N) 71.5 mg, Weight 12.4% wt loss on drying RTvac dry Change 5 hours DSC 150° C. (broad endo, min) at 50 mTorr ^(b)(crimped) 256° C. (endo, onset) with concurrent decomp (Form N) 43 mgXRPD unknown + Form N 42° C. vac dry 22 hours at 20 mTorr ^(b) (Form A)1.5 g, 9 mL Initial 0.9 g nitromethane, Recovery slurry 4 days, Weight0.4% wt loss on drying vac filter with Change acetone wash, PLMplaty/bladed particles (B/E) RT vac dry HR XRPD Form B + peaks ^(c) 6hours at 50 mTorr ^(b) 1.6 g (estimated) Initial 1.3 g 9 mL Recoverynitromethane, PLM rectangular plates/prisms slurry 5 days, (B/E) vacfilter with HR XRPD Form N nitromethane wash (Form N) 94,0 mg, Weight0.7% wt loss on drying RT vac dry Change 16.5 hours PLM rectangularplates/prisms at 20 mTorr ^(b) (B/E) HR XRPD Form N + weak peak at 9.1°2θ ¹H-NMR consistent with structure ⅓ mole nitromethane (Form N) 308.3mg, Weight 0.7% wt gain on drying ^(e) RT vac dry Change 5 days PLMrectangular plates/prisms at 5 mTorr^(d) (B/E) HR XRPD Form N + weakpeak at 9.1 °2θ DSC (open) 161° C. (broad endo, min) with shoulder at148° C. 259° C. (endo, onset) with concurrent decomp TGA 4.9% wt loss130-160° C. (equates to ~0.5 mole nitromethane) ¹H-NMR consistent withstructure ⅓ mole nitromethane 152.4 mg, Initial 97.7 mg 1 mL Recoveryacetonitrile, PLM rectangular plates/prisms brief vortex, (B/E) ^(f)slurry 1 hour, XRPD pattern T vac filter with Initial 270.4 mgacetonitrile Recovery wash/dry PLM rectangular plates/prisms severalminutes (B/E) ^(f) 309.5 mg, HR XRPD Forms C + A + peaks ^(g) 4 mLwater, brief vortex, slurry 24.5 hours, vac filter with water wash/ dry1.5 hours ^(a) Form N; additional weak peaks are present in the XRPDpattern at approximately 8.37, 11.37, 13.10, 16.23, and 21.86 °2θ. ^(b)Vacuum pressure from in-line gauge for vacuum system. ^(c) Additionalpeaks in the XRPD pattern present in XRPD pattern of Form A. ^(d)Samplestored in covered container at RT for 1 day prior to drying. Sampledried in stand-alone oven; vacuum pressure for oven measured by McLeodgauge. ^(e) 0.1% wt gain since check at 3 days. ^(f) Particles appearedunchanged by solvent. ^(g) Additional peaks in the XRPD pattern presentin XRPD pattern of Form N.

Example 13 Solubility Studies

The ambient temperature solubility data for the Compound I Form A aresummarized in Table 15. The solids exhibited apparent solubilities ofwell over 100 mg/ml for dimethylformamide (DMF), dichloromethane (DCM)and 2,2,2-trifluoroethanol (TFE). The material exhibited moderatesolubility (e.g., >10 mg/ml) in the majority of solvent and solventcombinations tested. The only exception was isopropanol (IPA) at 4.6mg/mL. Some solubility data was obtained on multiple samples aspresented in Table 15 below.

TABLE 15 Solubility data for Compound I, Form A Results ResultsSolvents^(a) [mg/ml] Solvents^(b) [mg/ml] Acetone 22.4, 28.2 (1:0.2)16.3 Ethanol:TFE (0.5:0.1) 38   (1.5:0.8) Ethyl 8.9 Acetone:DCMAcetate:Acetone (1:0.2) Acetonitrile 17.9 (1.5:1) 8.0^(d) (ACN):TFEHeptane:DCM 2-Butanone (MEK) 12.5 Isopropanol (IPA) 4.6 (1:0.1)2- 20.6(1:1)2- 11.5^(d) Butanone:TFE Propanol:Acetone Chloroform 26.5 (4.5:3)Isopropyl 6.8 Ether:Ethanol Dichloromethane 135.3, (1.5:1) 8.7^(d) (DCM)280 Methanol:TFE Dimethylformamide 248.5  Nitromethane 23.6 (DMF)(1:0.5) 13.3 2,2,2- 158.9 Dioxane:Acetone trifluoroethanol (TFE) Ethanol(EtOH) 23.5 (0.1:0.1) 97 TFE:DCM (1:1) Ethanol/IPA 28^(c ) (1:0.1) 21.4Toluene:TFE (l:3) Ethanol/IPA 10^(c ) (1.5:0.5) 12.4 Water:DCM ^(a)Ratioof solvents based on volume, in milliliters. ^(b)Solubility assessmentperformed at ambient temperature, unless otherwise noted. Reportedvalues are less than or equal to the actual solubility of Compound I ineach test solution based on visual observation and therefore areapproximate. ^(c)Experiment performed on hot plate set to 70° C.^(d)Experiment performed on hot plate set to 60° C.

Example 14 Polymorph Screen

A series of solvent-based experiments were set up utilizing slurry,evaporation, crash precipitation, and vapor diffusion techniques. Thesamples were prepared with Compound I Form A and the experimentalresults are summarized in Tables 16 and 17.

In one embodiment, experiments using solvents from the crystallizationprocesses for Forms A and B resulted in characterization of selectedsolids recovered from these experiments that displayed unique XRPDpatterns designated as Forms A to E, H, and J, and are described below.Several unique XRPD patterns were also obtained from other experimentalconditions, including an x-ray amorphous solid. No furthercharacterization of these solids was performed. A summary of exemplaryXRPD results for crystallization experiments are presented in Table 16.

TABLE 16 Summary of XRPD Results for Crystallization Experiments SolventConditions Habit/Description XRPD Result Dichloromethane fastevaporation clear film Form A (w/disorder) (DCM) Acetone fastevaporation white flakes Form B + peaks (Form A) (1.5:1) fastevaporation opaque film x-ray amorph MeOH:TFE (1:0.2) fast evaporationfilm on vial walls x-ray amorph EtOH:TFE (1:1) fast evaporation opaquefilm Form A (w/disorder) IPA:Acetone (1.5:0.8) fast evaporationblades/needles on Form A (w/disorder) EtOAc:Acetone vial walls (1.5:1)fast evaporation sm needles on vial wall Form A (w/disorder) Heptane:DCM(1.5:0.5) fast evaporation white solids, x-ray amorphous Water:DCMagglomerates (1:0.1) fast evaporation flakes B + A MEK:TFE (1:0.2) fastevaporation — — ACN:TFE (1:0.1) fast evaporation Agglomerate solids FormA Toluene:TFE 2,2,2- fast evaporation clear film similar to Form ETrifluoroethanol (TFE) (1:0.5) fast evaporation long needles, asperitesForm A Dioxane:Acetone (0.5:0.1) fast evaporation Agglomerate platesForm B + peaks (Form Acetone:DCM A) (0.1:0.1) fast evaporation clearfilm x-ray amorph TFE:DCM Methyl ethyl ketone slow evaporation — Form Jsimilar to Form (MEK) D Dimethylformamide slow evaporation no solids —(DMF) (4.5:3) slow evaporation thin film with B/E similar to Form A +Isopropyl ether:EtOH peaks Chloroform Slurry, vac dried, 60° C. Driedsolids Form H (1:3) Acetone/water −5° C., 1 day seeded with Dried solidsForm C + peaks (Form Form C + peaks (Form A) A) (1:3) Acetone/water −5°C., 41 days seeded with Dried solids Form C Form C + peaks (Form A)

A summary of exemplary XRPD results for vapor diffusion experiments arepresented in Table 17.

TABLE 17 Summary of XRPD Results for Vapor Diffusion Experiments Habit/Solvent^(a) Conditions Description^(b) XRPD Result Dichloro- HeptaneAgglomerate flakes Form A methane Water white solids precipitate, —undefined habit Methanol no solids — Heptane Agglomerate blades with B/EForm B (w/ disorder) Acetone Water white solids, undefined habit Form Awith B/E (3.5:1) Heptane no solids — Isopropyl Water white solidsprecipitate, — alcohol:Tri- undefined habit fluoro- MethanolAgglomerated needles/blades x-ray ethanol with B/E amorphous + peaksEthanol Heptane white solids precipitate, — Water undefined habit —Methanol — Trifluoro- Heptane no solids — ethanol Water white solidsprecipitate, — Methanol undefined habit — ^(a)Solvent ratios inparenthesis on volume basis, unless otherwise noted ^(b)B:birefringence, E: extinction (E) under cross polars

Example 15 Composition/Formulation

This example illustrates various components present in a representativeformulation containing Compound I according to the present disclosure,which was formulated as a bulk solution batch using the following steps:(a) preparing a Compound I solid form; (b) preparing a compoundingsolution comprising tert-butyl alcohol and water; (c) combining CompoundI solid form and the compounding solution to form a mixture; (d) addingpovidone to the mixture; (e) adjusting the pH of the mixture by addinghydrochloric acid solution, resulting in a formulated solution; (f)performing sterile filtration of the formulated solution; and (g)lyophilizing the formulated solution under aseptic conditions, to yielda final composition comprising Compound I. The steps are detailed inTable 18 below.

TABLE 18 Various components of the bulk solution Quantity per Ref toQuality Component Function 51 L Batch Standard Compound I Activepharmaceutical 204 g Internal ingredient Povidone Excipient 408 g USP0.1N hydro- pH adjustment 510 mL NF/EP chloric acid Nitrogen Processingagent and N/A NF/EP inert atmosphere for vial Headspace Water forProcessing agent 22.3 kg USP/EP Injection* Tert-butyl Processing agent26.1 kg ACS alcohol* *Removed during lyophilization

Example 16 Preparation of Lyophilate

Preparation of Compounding Solution

Following preparation and sterilization of components (stoppers andvials) of equipment needed, all processing equipment was inspected toassure it was free from residual rinse water. Vessel 1, a 20 gallon,jacketed, stainless steel vessel, was purged with nitrogen NF/EP. Therequired amount of tert-butyl alcohol was added to Vessel 1. Thetemperature of the tert-butyl alcohol and compounding vessel wereadjusted to 28 to 32° C. in advance to maintain this raw material as afree-flowing liquid. Following the addition of tert-butyl alcohol andinitiation of mixing, the required amount of water for injection (WFI)was added and the solution was mixed to completeness for 10±2 minutes,to result in a final compounding solution of 56 L. A portion (25%) ofthe compounding solution was transferred to a second, smaller, jacketed,stainless steel vessel (Vessel 2) for use in subsequent compoundingsteps. Both vessels were temperature controlled at 28 to 32° C., andVessel 1 was maintained with a nitrogen NF/EP overlay.

Preparation of Formulated Bulk Solution

Compound I solid form drug substance was weighed in an isolator and thentransferred directly to the compounding solution tank (Vessel 1) by wayof a single-use, disposable isolator transfer bag, to form a drugsubstance solution. The transfer bag was rinsed 3 times with a portionof the compounding solution from Vessel 2 and each rinse was added tothe compounding solution tank.

The drug substance solution was mixed for 30±5 minutes at 28 to 32° C.Following dissolution of amorphous Compound I, the specified amount ofpovidone, USP, was added to the compounding vessel. The weighingcontainer was rinsed once with a portion of the compounding solution andthe rinse was transferred to the compounding tank that was mixed for20±5 minutes at 28 to 32° C. to dissolve the povidone.

The pH of the bulk solution was adjusted with a predetermined amount of0.1 N HCl solution and was mixed for 10±2 minutes at 28 to 32° C. toform a formulated bulk solution. The formulated bulk solution wassampled and the apparent pH was verified to be between 3.6 and 4.0. TheQS volume of compounding solution required to achieve the calculatedtarget weight was transferred from Vessel 2 to Vessel 1. The formulatedbulk solution was mixed for 10±2 minutes at 28 to 32° C. and thensampled for quality control (QC) testing, including appearance, assay,density, pH, and bioburden. The compounding tank was sealed, and thetemperature was maintained at 28 to 32° C. until sterile filtration.

Sterile Filtration of Formulated Bulk Solution

The compounding tank containing the formulated bulk solution was movedfrom the Class 100,000 compounding suite to an anteroom adjacent to theClass 10,000 filling suite. The formulated bulk solution was transferredvia a 3/8″ stainless-steel braided Teflon® hose passed through a port inthe wall of the sterile filling suite to the filling suite by overpressurization with sterile nitrogen, NF/EP. The formulated bulksolution was first clarified through a Millipore Opticap® filter (0.22μm Durapore® membrane) and then was sterilized by filtration through afilter assembly located within the aseptic core containing 2 MilliporeMillipak® 0.22 μm Durapore® filters in series, into a sterile receivingvessel. The integrity of the product sterilizing filters was tested forpressure and flow pre- and post-filtration using Isopropyl Water(IPA)/Water (60%/40%) as the wetting solution. The minimum pressure holdvalue was 10 psi prior to filtration, and the maximum flow is 1.3 mL/minat 12 psi after filtration. The sterile-filtered formulated bulksolution was sampled for QC testing, including appearance, assay,density, and pH.

Aseptic Filling of Vials for Drug Product

Aseptic filling and stoppering of the sterile vials occurred under Class100 conditions using an automated TL filling line. Process controlsincluded defined weight checks of vials to verify accurate fill volumethroughout the filling operation.

Immediately following filling of each vial, a sterile lyophilizationstopper was partially seated in the vial and each tray of filled vialswas moved to the loading area for the lyophilizer within the Class 100aseptic area. Trays were immediately loaded onto precooled shelves inthe lyophilizer.

Lyophilization

Vials containing compositions were lyophilized under aseptic conditionsusing a preprogrammed lyophilization cycle. A summary of thelyophilization cycle process and controls is provided in Table 19.

TABLE 19 Lyophilization Process and Controls Lyophilizer ProgramSegments Process Set Points Controls Limits 1-4: Chamber loading andLoad vials into chamber Shelf temperature: 0 ± 3° C. freezing Ramp shelftemperature down Shelf temperature: −40° C. to −45 ± 3° C. Productthermocouples: ≦−40° C. 5: Hold Hold at temperature for 2 ± 0.5 hoursProduct thermocouples: ≦−40° C. 6: Evacuate chamber Evacuate chambervacuum to 100-200 Chamber pressure: 100-200 μm μm 7-8: Ramp temperatureRamp shelf temperature up to −20 ± Shelf temperature: ≧−23° C. and hold3° C. over 3 hours (~8° C./hour) Product thermocouples: ≧−23° C. Holdfor 2 ± 0.5 hours 9: Ramp temperature; Ramp shelf temperature up to 0 ±3° Shelf temperature: 0 ± 3° C. nitrogen sweep C. over 2 ± 0.5 hours(~10° C./hour) Product thermocouples: ≧−3° C. Nitrogen sweep at 135 μmChamber pressure: 100-200 μm 10: Ramp temperature Ramp shelf temperatureup to 33 ± Shelf temperature: 33 ± 3° C. and hold 3° C. over 6 hours(~6° C./hour) Product thermocouples: ≧27° C. Hold at temperature for 2hours Chamber pressure: 100-200 μm 11: Terminal drying¹ Pull chamberpressure to ≦100 μm Shelf temperature: 33 ± 3° C. Hold for 16 ± 1 hoursProduct thermocouples: ≧27° C. Chamber pressure: <100 μm 12: End cycle;stopper; Increase chamber pressure to 14 to 15 Shelf temperature: 5 ± 3°C. hold for unloading² psia with Nitrogen NF/EP Product thermocouples:5° C. Ramp shelf temperature down to 5 ± Chamber pressure: 15 psia 3° C.Seat stoppers 13: Product unloading Ramp shelf temperature up to 20 ±Product thermocouples: ≧15° C. 3° C. Chamber pressure: 14 psia Openchamber and unload ¹Total terminal drying time, including initial 2 hourhold, is 18 ± 1 hours ²The shelf is cooled to 5 ± 3° C. only if it isnecessary to hold the product for an extended time prior to unloading.

In one embodiment, an additional step after the secondary dryingfollowing step 11 (Table 19) includes drying the vials at thetemperature of 50° C. up to 24 hours at the pressure of 50 μm Hg. Inanother embodiment, an additional step includes drying the vials at thetemperature of 50° C. up to 48 hours at the pressure of 50 μm Hg.

In another embodiment, an additional step after the secondary dryingfollowing step 11 (Table 19) includes drying the vials at thetemperature of 60° C. up to 3 hours at the pressure of 100 μm Hg. In yetanother embodiment, an additional step includes drying the vials at thetemperature of 60° C. up to 6 hours at the pressure of 100 μm Hg. Inanother embodiment, an additional step includes drying the vials at thetemperature of 60° C. up to 12 hours at the pressure of 100 μm Hg. Inanother embodiment, an additional step includes drying the vials at thetemperature of 60° C. up to 24 hours at the pressure of 100 μm Hg. Inanother embodiment, an additional step includes drying the vials at thetemperature of 60° C. up to 48 hours at the pressure of 100 μm Hg.

In another embodiment, an additional step after the secondary dryingfollowing step 11 (Table 19) includes drying the vials at thetemperature of 70° C. up to 24 hours at the pressure of 25 mm Hg. Inanother embodiment, an additional step includes drying the vials at thetemperature of 70° C. up to 48 hours at the pressure of 25 mm Hg.

Following completion of the cycle (Segment 12), the vials werebackfilled with sterile nitrogen, NF/EP, at atmospheric pressure and thestoppers were completely seated prior to opening the lyophilizerchamber. The trays were unloaded and transferred to the sealing area.

Vials containing compositions were sealed immediately followingunloading from the lyophilization chamber. Each seal was imprinted withthe Composition lot number using a video jet printer incorporated intothe automated sealing line. Seal inspection is performed every 15minutes during the sealing operation.

Following sealing operations, Compound I composition vials wereinspected, labeled and packaged and appropriate process validationand/or Evaluation was subsequently performed.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the disclosure, described herein. The scope of thepresent disclosure is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The disclosure includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Thedisclosure includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process. Furthermore, it is to be understood that thedisclosure encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim. For example, any claim that is dependent on another claim can bemodified to include one or more limitations found in any other claimthat is dependent on the same base claim.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the disclosure, or aspects ofthe disclosure, is/are referred to as comprising particular elements,features, etc., certain embodiments of the disclosure or aspects of thedisclosure consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is noted that the term“comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of skill in the art, values that areexpressed as ranges can assume any specific value or subrange within thestated ranges in different embodiments of the disclosure, to the tenthof the unit of the lower limit of the range, unless the context clearlydictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present disclosure that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of skill in the art, they may be excluded evenif the exclusion is not set forth explicitly herein. Any particularembodiment of the compositions of the disclosure (e.g., any targetingmoiety, any disease, disorder, and/or condition, any linking agent, anymethod of administration, any therapeutic application, etc.) can beexcluded from any one or more claims, for any reason, whether or notrelated to the existence of prior art.

Publications discussed above and throughout the text are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The invention claimed is:
 1. A crystalline form C of romidepsincomprising XRPD characteristic peaks at about 8.28 2θ, 11.45 2θ, 12.192θ, and 21.13 2θ.
 2. A pharmaceutical composition comprising thecrystalline form C of romidepsin of claim 1 and a pharmaceuticallyacceptable excipient.